Method and apparatus for analyzing acetone in breath

ABSTRACT

Methods and devices are provided for analyzing acetone in breath. One such method comprises disposing a reactant in a reaction zone within the breath analysis device, wherein the reactant comprises a primary amine disposed on a surface, and wherein the reaction zone has an optical characteristic that is at a reference level. It also comprises pre-storing a liquid nitroprusside solution within the breath analysis device separately from the reactant. The method further comprises using the breath analysis device to cause the breath to contact the reactant in the reaction zone so that the acetone in the breath reacts with the reactant to form a reaction product and, after the reaction product has been formed, using the breath analysis device to cause the nitroprusside solution to contact and react with the reaction product and to facilitate a change in the optical characteristic of the reaction zone relative to the reference level.

FIELD OF THE INVENTION

The present invention relates generally to methods, apparatus andtechnology for analyzing acetone in the breath of a living thing,preferably a human.

BACKGROUND OF THE INVENTION

Ketone bodies include acetoacetic acid (beta-ketobutyric acid, diaceticacid), beta-hydroxybutyric acid, and acetone. For purposes of gasanalysis, acetone is the most important of these, given its relativelyhigh vapor pressure. The advantages of analyzing acetone, or otherketone bodies or ketones, in breath are well recognized and have beenfor decades. Under normal, unstressed physiological circumstances, forexample, the body metabolizes primarily carbohydrates. When normalcarbohydrate metabolism is impaired, however, the body begins tometabolize fats or fatty acids. During fat metabolism, ketone bodiesincluding acetone are produced as intermediaries and begin to accumulatein body fluids such as blood and urine. As the accumulated ketone bodiesare transported in the blood, they become involved the gas exchange inthe alveolar spaces of the lungs. This is particularly true of acetone,given its low molecular weight and vapor pressure. As a result of thispulmonary transport, the acetone appears in the breath, includingexpired breath, where it can be analyzed. The term “analyze” as usedherein is used in is common but broad sense to include such things asdetection of the presence of a chemical component. It also may includemeasurement of concentration or of properties or conditions of thecomponent.

The ability to analyze acetone and in some cases other ketone bodies orketones is important in health care. An accumulation of acetone orketone bodies is generally referred to as “ketosis” or “ketoacidosis.”These conditions can have toxic effects and in extreme cases can lead topermanent injury or death. Patients suffering from diabetes mellitus,for example, are susceptible to ketoacidosis.

During ketoacidosis, elevated levels of ketone bodies occur in bodyfluids, mainly in blood, urine and breath. The primary means for testingto identify these elevated ketone body levels has been through bloodand/or urine analysis.

A common and well known technique for analyzing ketone bodies in bloodand urine is the so-called “Legal test,” in which appropriately solublenitroprussides are used to react with and correspondingly detect certainketone bodies. Swinehart, Coordination Chem. Rev., 2(4), 386-403(December 1967) is often cited. It discloses a summary of workundertaken on the reaction of sodium nitroprusside with acetone andaceoacetic acid. He described a mechanism of reaction in which thesodium nitroprusside reacts at the site of the acidic hydrogen, andaction of the nitrosyl moiety of sodium nitroprusside.

U.S. Pat. No. 2,186,902, issued to Fortune on Jan. 9, 1940, disclosesthe use of nitroprusside wherein the reaction is carried out in thepresence of ammonia to develop and utilize color-based detectionfeatures. U.S. Pat. No. 2,509,140 discloses a nitroprusside reaction inurine wherein the reaction takes place in the presence of an aliphaticamino acid, i.e., glycine, and an alialkine material. U.S. Pat. No.2,900,253, issued to Smeby on Jun. 27, 2961, discloses a test methodthat use of dip sticks or swabs chemically treated with a nitroprussideto obtain a sample of the blood or urine and test for the presence ofketone bodies.

In contrast to ketone body testing in blood and urine, the use of breathfor ketone body analysis has been far more limited. Abbott Laboratories,in several issued patents, disclosed methods for detecting acetone inbreath using nitroprusside and a primary amine. See, e.g., U.S. Pat.Nos. 4,970,172, 5,071,769 and 5,174,959. As reported therein, Abbottused a tertiary amine with a pH of 4.5 and a primary amine without anyadjustment pH. Methods disclosed therein used a developer solution with0.5% diethanolamine in 25% dimethylsulfoxide and 75% methanol. While theoverall concept reportedly was able to detect acetone in breath, it hada long manufacturing time (four days), a long reaction time (18minutes), a large consumable materials requirement with relativelyexpensive reagents packed inside, and was not adequately stable overlong periods of time. Although the device disclosed by Abbott reportedlywas operable, it was not commercially feasible.

A pronounced problem encountered in nitroprusside-based tests for ketonebodies in general lies in the instability of the nitroprusside. As isnoted in U.S. Pat. No. 2,990,253, for example, sodium nitroprusside isvery unstable in an aqueous, alkaline medium. Unfortunately, however,this is precisely the type of medium that is required to achieve thedesired reaction between the ketone (in that case sodium acetoacetate)and sodium nitroprusside.

The solution to this instability problem proposed and claimed in U.S.Pat. No. 2,990,253 involved preparation of a stick test using a two-stepapproach. The first step involved applying the nitroprusside to acarrier in an acidic aqueous medium, and drying. The second involveddipping the carrier into a non-aqueous solution of organic bases, suchas various amines, aminoalcohols or mixtures thereof, to achieve thealkalinity needed for the desired reaction with the ketone body. Theaqueous solution comprised sodium nitroprusside, glycine, monosodiumphosphate-monohydrate, disodium phosphate and sodium chloride. Thesecond step comprised using a secondary or tertiary amine oraminoalcohol or a mixture thereof in anhydrous ethanol or chloroform.

U.S. Pat. No. 4,147,514, issued to Magers et al. on Apr. 3, 1979,suggests addressing the instability problem by using nitroprusside incombination with at least one primary amine, and a metal salt. The metalsalt reportedly stabilized the nitroprusside in solution at alkaline pHranges, allowed for a single-dip production method, promoted ionizationof the ketone bodies, resulted in a shorted reaction time, andstabilized the resulting chromophoric complex.

Abbott, e.g., in the aforementioned patents, noted that the colorcomplex is unstable because nitroprusside decomposes rapidly in alkalinesolutions, and that nitroprusside salts are subject to decomposition inthe presence of moisture and high pH. According to Abbott, theselimitations have led to numerous attempts to stabilize the color complexby utilizing mixtures of nitroprussides and amines or amino acids incombination with a variety of buffers, metal salts, organic salts,organic stabilizers and polymers. Abbott then provided a summary ofefforts by various parties as reported in various issued patents aimedto addressing this problem. Abbott proposed the use of a first solidmatrix material to which a nitroprusside salt is coupled, and a secondsolid matrix material to which an amine is covalently bound. Abbot alsoproposed the addition of magnesium or calcium salts to promote chelateformation and to stabilize the color products and enhance the kineticsof the reaction between the carbonyl compound, the amine and thenitroprusside.

Notwithstanding these efforts, the stability of the nitroprusside-basedtest regimes as described herein above have persisted and have limitedthe use of such regimes for the analysis of ketone bodies in breath andother gases. Abbott, for example, apparently never advanced its breathacetone product development to commercial fruition.

SUMMARY OF THE INVENTION

The present inventions according to its various aspects provide methodsand apparatus for detecting or otherwise analyzing ketone bodies,particularly acetone, in breath using primary amines and nitroprusside,which methods and apparatus provide enhanced stability, enhancedresponse time, and/or enhanced chromic qualities.

In accordance with one aspect of the invention, a method for is providedfor analyzing acetone in breath. The method comprises providing acavity, locating within the cavity a primary amine disposed on a firstsurface, and locating within the cavity a nitroprusside on a secondsurface distinct from the first surface. The nitroprusside is coupled tothe second surface using a coupling agent comprising an anion exchangeresin in an acidic environment, wherein the acid environment comprisesan acid having a vapor pressure of less than about 1 atmosphere at 22 Cwhen in its 99% pure form. The primary amine on the first surface andthe nitroprusside on the second surface comprising cavity contentshaving a reference optical property. The method further comprisescausing the breath to move into the cavity so that it contacts theprimary amine and the nitroprusside to cause or facilitate a change inan optical property of the cavity contents relative to the referenceoptical property. It also comprises analyzing the breath for thepresence of the acetone using the change in the optical property.

The cavity preferably but optionally comprises an elongated and enclosedchannel. It preferably contains the primary amine on the first surfaceand the nitroprusside on the second surface in the form of a packed bed.

The breath typically comprises a moisture content, and the method mayfurther comprise pretreating the breath prior to causing the breath tocontact the primary amine to reduce the moisture content.

The primary amine preferably has a pKb of less than 5. A presentlypreferred primary amine comprises an amino silane.

The first surface preferably comprises a plurality of beads comprised ofsilica, quartz, aluminum oxide, alumino-silicates, silicon, copper, tinoxide, talc, inorganic oxides, or combinations thereof.

The nitroprusside preferably comprises sodium nitroprusside. The secondsurface preferably comprises a plurality of beads comprised of silica,quartz, aluminum oxide, alumino-silicates, silicon, copper, tin oxide,talc, inorganic oxides, or combinations thereof. The coupling agent maycomprise a tertiary amine, and preferably comprises diethylaminopropyltrimethoxysilane. The acidic environment preferably comprises sulfuricacid, and substantially excludes hydrochloric acid.

The reference optical property preferably comprises a reference colorand the change in optical property preferably comprises a change incolor with respect to the reference color. The cavity in presentlypreferred embodiments and methods has a linear dimension, the referenceoptical property preferably comprises a reference distance of thereference color along the linear dimension, and the change in opticalproperty preferably comprises a measured distance along the lineardimension of the change in color with respect to the reference color.The method may comprise introducing a developer solution into the cavityto facilitate the change in the optical property.

In accordance with another aspect of the invention, a method is providedfor analyzing acetone in breath. This method comprises providing acavity, locating within the cavity a primary amine disposed on a firstsurface, and locating within the cavity a nitroprusside on a secondneutral surface distinct from the first surface. The nitroprusside is ina dry state. The primary amine on the first surface and thenitroprusside on the second surface comprise cavity contents having areference optical property. The method also comprises causing the breathto move into the cavity so that it contacts the primary amine and thenitroprusside to cause or facilitate a change in an optical property ofthe cavity contents relative to the reference optical property, andanalyzing the breath for the presence of the acetone using the change inthe optical property.

Preferred features of the first aforementioned also apply to thismethod.

The nitroprusside on the second neutral surface may be attached to thesecond surface without use of a coupling agent. It may be adsorbed ordried onto the second neutral surface, or otherwise be disposed thereonwithout a chemical bond.

The nitroprusside on the second surface preferably is initially locatedfluidically separately from the primary amine on the first surface, andthe method comprises sequentially reacting the breath with the primaryamine to yield a reaction product, and then contacting the nitroprussidewith the reaction product. The method also may comprise introducing adeveloper solution into the cavity to facilitate the change in theoptical property.

In accordance with another aspect of the invention, a method is providedfor analyzing acetone in breath, wherein the method comprises providinga cavity, and locating within the cavity a primary amine disposed on afirst surface. The primary amine on the first surface comprises cavitycontents having a reference optical property. The method also comprisesproviding a nitroprusside in a nitroprusside solution initiallyseparated from the primary amine on the first surface, whereinconditions of the nitroprusside solution when separated from the primaryamine on the first surface are selected to stabilize the nitroprussiderelative to the reactivity of the nitroprusside in the cavity with theprimary amine. The method further comprises causing the breath to moveinto the cavity so that it contacts the primary amine to create aprimary amine reaction product, and causing the nitroprusside solutionto enter the cavity and the nitroprusside to react with at least one ofthe acetone and the primary amine reaction product, to cause orfacilitate a change in an optical property of the cavity contentsrelative to the reference optical property, and analyzing the breath forthe presence of the acetone using the change in the optical property.

The optional but preferred aspects of the invention noted herein abovealso may apply to this aspect of the invention. It is also preferablethat the providing of a nitroprusside in a nitroprusside solutioninitially separated from the primary amine on the first surfacecomprises preventing light from contacting the nitroprusside solution,and also may comprise limiting light from contacting the nitroprussidesolution sufficiently that the effectiveness of the nitroprusside inreacting with at least one of the acetone and the primary amine reactionproduct and the causing or facilitating of the change in an opticalproperty of the cavity contents relative to the reference opticalproperty are not impaired.

In accordance with yet another aspect of the invention, a method isprovided for analyzing acetone in breath. The method comprises preparinga surface upon which is disposed a primary amine and a nitroprusside.The preparation comprises disposing the primary amine and thenitroprusside in an acidic environment. The method also compriseslocating the surface upon which is disposed the primary amine and thenitroprusside within a cavity. The surface comprising the primary amineand the nitroprusside comprises a reference optical property. Thesurface preferably is in proximate contact with a solution. The methodfurther comprises causing the breath to move into the cavity so that itcontacts the primary amine and the nitroprusside to cause or facilitatea change in an optical property of the surface relative to the referenceoptical property. The method further comprises analyzing the breath forthe presence of the acetone using the change in the optical property.

Again, the optional but preferred features noted herein above also mayapply to this aspect of the invention.

The acidic environment according to this aspect of the inventionpreferably has a pH of between 0 and 6, and more preferably has a pH ofbetween 0.5 and 2.

The method also may comprise exposing the surface to a second solution,wherein the second solution comprises a base having a alkalinity atleast as strong as an alkalinity of the primary amine, and wherein thebase is present in a concentration sufficient to increase extent of thereaction and the change in the optical property of the surface beyondthe extent of the reaction and change in the optical property in theabsence of the second solution.

In various embodiments and method implementations of the inventionaccording to one or more of these aspects, use preferably is made ofrelatively higher concentrations of nitroprusside on the surface towhich it is immobilized or in solution. The higher concentrations drivethe kinetics to improve reaction time and test response time, and canimprove sensitivity. In addition, enhanced stability can be achieved incertain of these embodiments and implementations using such features as:(i) less volatile acids (e.g., H₂SO₄ instead of HCl), (ii) more acid(i.e., a lower pH), (iii) surfaces that are not inherently basic (e.g.,avoiding the use of tertiary amines for supporting the nitroprusside),and (iv) protecting the nitroprusside from light or other degradingradiations.

In addition, and in accordance with another aspect of the invention, acartridge is provided for containing the reagents, e.g., such as thosedescribed herein, and for practicing methods as described herein and thelike.

In presently preferred embodiments and method implementations accordingto these various aspects of the invention, one can achieve fastermanufacturing times (e.g., less than four hours), faster reaction times(e.g., less than six minutes), lower cost and/or greater stabilityrelatively to prior known and reported methods and devices. Thesefeatures or results also can be achieved in the form of relativelysmall, portable devices that are relatively inexpensive, easy to use andare amenable to field or patient home use.

In accordance with another aspect of the invention, a method is providedfor sensing acetone in breath using a breath analysis device. The methodcomprises disposing a reactant in a reaction zone within the breathanalysis device, wherein the reactant comprises a primary amine disposedon a surface, and wherein the reaction zone has an opticalcharacteristic that is at a reference level. It also comprisespre-storing a liquid nitroprusside solution within the breath analysisdevice separately from the reactant. The method further comprises usingthe breath analysis device to cause the breath to contact the reactantin the reaction zone so that the acetone in the breath reacts with thereactant to form a reaction product and, after the reaction product hasbeen formed, using the breath analysis device to cause the nitroprussidesolution to contact and react with the reaction product and tofacilitate a change in the optical characteristic of the reaction zonerelative to the reference level. The method also comprises using thebreath analysis device to detect the change in the opticalcharacteristic to sense the acetone in the breath.

Optionally, causing the nitroprusside solution to contact the reactionproduct has an optical characteristic that is at a second referencelevel. Then, causing the nitroprusside solution to react with thereaction product facilitates a change in the optical characteristic ofthe reaction zone relative to the second reference level.

Optionally but preferably, the surface comprises a silica gel, and morepreferably a plurality of silica gel beads, preferably having a sizedistribution between 100 and 270 mesh.

It is also preferred that the disposing of the reactant in the reactionzone comprises maintaining the reactant in an alkaline environment. Thedisposing of the reactant in the reaction zone also may comprisemaintaining the reactant in the absence of volatile acid, or quenchingthe primary amine with a non-volatile acid. An example of a non-volatileacid would be sulfuric acid.

The pre-storing of the liquid nitroprusside solution within the breathanalysis device separately from the reactant preferably comprisesproviding the liquid nitroprusside solution to consist essentially of anon-alkaline solution. It also may and preferably does compriseproviding the liquid nitroprusside solution in the absence of asubstance with a base dissociation constant less than 6.

The pre-storing of the liquid nitroprusside solution also may comprisequenching the liquid nitroprusside solution with a non-volatile acid,such as sulfuric acid. This quenching preferably is undertaken so thatthe liquid nitroprusside solution has a pH of less than 8, and morepreferably below 7.

It is also preferably that the liquid nitroprusside solution be storedin the absence of ambient light.

In accordance with still another aspect of the invention, a method isprovided for sensing acetone in breath using a breath analysis device.The method comprises disposing a reactant in a reaction zone within thebreath analysis device, wherein the reactant comprises a primary aminedisposed on a surface, and wherein the reaction zone has an opticalcharacteristic that is at a reference level, pre-storing a nitroprussidein the breath analysis device in a non-alkaline environment, and usingthe breath analysis device to cause the breath to contact the reactantin the reaction zone so that the acetone in the breath reacts with theprimary amine to form a reaction product. After the reaction product hasbeen formed, the method further comprises using the breath analysisdevice to cause the nitroprusside to contact and react with the reactionproduct and to facilitate a change in the optical characteristic of thereaction zone relative to the reference level, and using the breathanalysis device to detect the change in the optical characteristic andto sense the acetone in the breath.

The disposing of the reactant in the reaction zone within the breathanalysis device optionally but preferably comprises quenching theprimary amine on the surface with a non-volatile acid so that theprimary amine on the surface has a pH that is less than 8, and morepreferably less than 7. An example of a suitable non-volatile acid issulfuric acid.

The pre-storing of the nitroprusside in the breath analysis device in anon-alkaline environment preferably comprises pre-storing thenitroprusside separately from the reactant prior to the causing of thenitroprusside to contact the primary amine reaction product, preferablyusing a gas-tight barrier.

The pre-storing of the nitroprusside in the breath analysis device in anon-alkaline environment preferably comprises pre-storing thenitroprusside in the absence of a substance with a base dissociationconstant less than 6.

The pre-storing of the nitroprusside in the breath analysis device in anon-alkaline environment preferably comprises quenching thenitroprusside with a non-volatile acid, such as sulfuric acid. Thepre-storing of the nitroprusside in the breath analysis device in anon-alkaline environment also preferably comprises quenching thenitroprusside with a non-volatile acid so that the environment has a pHis below 8, and more preferably below 7.

In one embodiment, the primary amine is disposed on a first surface,e.g., silica gel beads, the nitroprusside is disposed on a secondsurface other than the first surface, such as silica gel beads havingthe nitroprusside but not the primary amine, and the first and secondsurfaces, e.g., the two sets of beads, are intermingled prior to thecausing of the nitroprusside to contact the reaction product. In thissetting, the causing of the nitroprusside to contact and react with thereaction product preferably comprises dispensing a liquid developersolution to contact the first and second surfaces, or intermingledbeads.

In another alternate embodiment the nitroprusside is coupled to thereactant prior to the causing of the nitroprusside to contact theprimary amine product. The reactant and the nitroprusside are disposedin the non-alkaline environment, and the causing of the nitroprusside tocontact and react with the reaction product comprises dispensing aliquid developer solution to contact the nitroprusside and the reactionproduct.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate a presently preferred embodimentsand methods of the invention and, together with the general descriptiongiven above and the detailed description of the preferred embodimentsand methods given below, serve to explain the principles of theinvention. Of the drawings:

FIG. 1 shows a composite illustration of a device and disposablecartridge used in detecting colorimetric changes from reactions withbreath analytes.

FIG. 2 shows an example of a breath collection bag with integrated flowmeasurement capabilities.

FIG. 3 demonstrates an example of an indirect breath collectionperformed by a breath input.

FIG. 4 depicts a general layout for an optical sensing subsystemconfiguration.

FIG. 5 depicts a general layout for an optical sensing subsystemconfiguration from a top-view.

FIG. 6 depicts one pneumatic handler suitable for high quality breathgas measurements.

FIG. 7 shows one approach to component reduction using a specializedball valve.

FIG. 8 shows an exemplary reaction initiator based on a needle.

FIG. 9 shows a cartridge insertion into a base unit that makes use of alinear actuator.

FIG. 10 shows the details of an embodiment of a sliding mechanism inrelation to a sensor cartridge.

FIG. 11 shows an example of a breath gas analyzer column based on TenaxTA.

FIG. 12 displays an example of a substrate sheet that can be pressedinto retention disks.

FIG. 13 shows an exemplary general schematic of cartridge design.

FIG. 14 shows one alternative to the retainer (130) of FIG. 13 forcontaining reactive particles.

FIG. 15 shows four embodiments of a piercable foil ampoule.

FIG. 16 shows certain embodiments of a piercable ampoule.

FIG. 17 shows embodiments of a piercable can.

FIG. 18 shows different dry reagents packed into a single column.

FIG. 19 shows another set of stacked dry reagents packed into a singlecolumn.

FIG. 20 illustrates reagents being held in place using compressible,porous media.

FIG. 21 shows an example of how a liquid reagent can be immobilized ontoa cartridge and how it can be released at the time of reaction.

FIG. 22 demonstrates another embodiment of how a liquid reagent can beimmobilized onto a cartridge and how it can be released at the time ofreaction.

FIG. 23 illustrates an example of a multi-liquid cartridge.

FIG. 24 illustrates another example of a multi-liquid cartridge.

FIG. 25 shows some cartridge designs that enable multiuse applications.

FIG. 26 shows an embodiment of a cartridge design.

FIG. 27 shows a depiction of the flow path after the liquid seals havebeen broken and a liquid seal is formed.

FIG. 28 shows an embodiment of a cartridge with a developer solution.

FIG. 29 shows an embodiment of a breath sampling loop based on multiplebreath exhalations into a base unit.

FIG. 30 shows an embodiment of a breath measurement system with thedeveloper solution inside a replaceable container in the base unitinstead of in disposable cartridges.

FIG. 31 shows a reaction scheme for analyzing acetone in breath.

FIG. 32 shows a reaction scheme for analyzing acetone in breath.

FIG. 33 shows a reaction scheme for analyzing acetone in breath.

FIG. 34 shows a reaction scheme for analyzing acetone in breath.

FIG. 35 shows a chemical structure of nitroprusside.

FIG. 36 shows optical characteristics of the reaction zone.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS AND METHODS OF THEINVENTION

Reference will now be made in detail to the presently preferredembodiments and methods or method implementations of the invention asdescribed herein below and as illustrated in the accompanying drawings,in which like reference characters designate like or corresponding partsthroughout the drawings. It should be noted, however, that the inventionin its broader aspects is not limited to the specific details,representative devices and methods, and illustrative examples shown anddescribed in this section in connection with the preferred embodimentsand methods. The invention according to its various aspects isparticularly pointed out and distinctly claimed in the attached claimsread in view of this specification, and appropriate equivalents.

To better appreciate and illustrate the various aspects of the inventionas set forth herein, some background on the general reaction of ketonebodies using a nitroprusside color reaction test regime is useful andinstructive. Reactions of nitroprusside to analyze ketone bodies, e.g.,the Legal test, generally take place under the following reactionregime. There are two primary reactants are used: (1) one or moreprimary amines, and (2) the nitroprusside. In preferred regimes knownand reported publicly, such as those disclosed by Abbott, each of thesereactants is immobilized on a separate surface, typically usingsolid-phase beads, although it is possible to put both reactants on thesame surface. The surfaces are usually in the same reaction zone.

The reaction dynamics involve the following steps:

1. Acetone is introduced into the reaction zone. The acetone or otherketone body contacts the primary amine or amines on the first surface,whereupon the acetone reacts with the primary amine(s) to form a Schiffbase.

2. A solvent or “developer solution” is introduced into the reactionzone. The solvent solubilizes the Schiff base and the nitroprusside.

3. The Schiff base reacts with the nitroprusside to yield a colorproduct. This reaction causes a color change, from reactant to product.The specific color indicates the presence of the reactant, i.e.,acetone. The extent of advance through the reaction zone indicates theconcentration of the acetone in the breath sample.

While a number of nitroprusside based ketone detection urinalysis andblood based methods have been described in the prior art, none of theseis believed to be capable of detecting acetone, especially not as it ispresent in breath. While reactions with acetone have been reported, whatthey are appears to be occurring is a reaction with ketone bodiesacetoacetic acid and/or beta-hydroxybutyrate (b-HBA). The water presentin urine and blood inhibits visible color change from acetone at levelspresent in the body. Even the humidity in normal breath is sufficient toprevent color formation in a reaction between acetone, a primary amineand nitroprusside.

Given the much slower and weaker reaction of acetone with primary aminesand nitroprusside, a variety of changes to the chemistry and reactionmethods must be made to enable detection at levels present in breath.First, all or essentially all traces of water must be removed from thebreath sample. This can be accomplished through a variety of dryingmethods, including but not limited to the use of desiccants. Preferreddesiccants include ascarite, sodium hydroxide, and calcium chloride.Large calcium chloride particles do not provide sufficient surface areato extract humidity in a small chamber. Smaller CaCl₂ particles arepreferred, between about 20 and about 200 mesh, between about 30 and 100mesh, between about 35 and 60 mesh.

Next, one must have a method for concentrating the acetone from breathup to detectable levels. This can be accomplished by passing the breathsample across a packed bed or similar high surface area medium withaffinity for acetone. In preferred embodiments and methods according tothe present invention, the component in the packed bed with affinity foracetone is the primary amine used to form the Schiff base with acetone.In order to force the breath through the packed bed, it is contained insuch a fashion that a gas tight seal can be made and the sample has onlyone direction to flow. This can be accomplished by enclosing theaffinity reagents for acetone in a cavity.

Flow rates scan be optimized in order to: 1) remove sufficient watercontent and 2) concentrate the acetone on a sufficiently small region ofthe packed bed to create the concentration necessary for a visible colorchange. Optimal flow rates depend on the amount and size of thedesiccant and the dimensions of the packed bed. The flow rate preferablyis sufficient to flush out dead volume and provide a good averageacetone level in breath in less than 10 min, or more preferably in lessthan 5 min. Further, the flow rate must be slow enough that it does notuse excessive desiccant or reagent, and does not require samplecollection bags larger than 10 L, or more preferably larger than 1 L.Preferred flow rates are between about 10 and about 1000 mL/min, betweenabout 50 and about 250 mL/min, between about 100 and 150 mL/min. Flowrates also preferably are constant, within plus or minus 30%, or morepreferably 10%, or even more preferably, 1% of the target flow rate.This will provide consistent, repeatable color formation.

The reaction also should be more alkaline than is commonly used inurinalysis and blood tests for acetone. The alkalinity of the reactionshould match the pKa of the conjugate acid of the primary amine in orderto have rapid and sensitive reaction kinetics. Because nitroprussidedegrades in alkaline environments, in presently preferred embodimentsand methods it is either stored in a separate location from the primaryamine or the pH is adjusted in a separate step. In either case, aseparate developer solution is used to solvate the nitroprusside and/orto modify the pH of the reaction.

Each of the foregoing changes significantly improves the ability todetect acetone, especially at the concentrations at which acetone ispresent in breath. In contrast, none of these features is required fordetection of acetoacetic acid and b-HBA in blood and urine. But becauseknown urinalysis and blood testing methods do not incorporate theforegoing changes together, they are incapable of detecting acetone,especially at the concentrations present in breath.

Methods and devices according to the present invention preferably areimplemented using a breath analysis device that is configured to carryout the methods as generally described herein and their equivalents.Although not necessarily limiting, U.S. Application No. 61/593,862 the('862 Application”), filed on Feb. 1, 2012 and commonly assigned to thepresent inventors' assignee, Invoy Technologies, LLC, provide variousdescriptions, illustrations and examples of devices and supportingmethods upon which preferred embodiments and method implementations ofthe present invention may be carried out. The '862 Application is herebyincorporated by reference as if fully set forth herein, and the readeris directed to that application for a description of details andalternate variations. It should be understood, however, that the presentinvention according to these aspects is not necessarily limited to suchspecific and illustrative devices and methods of that application.

Devices and methods according to the '862 Application can include orincorporate any or all of a base, a breath input, an insertion mechanismfor a cartridge, a sensing subsystem, a pneumatic handler, a reactioninitiator, a kinetic enhancer, a breath conditioner, a digitizer, or acartridge. Each of these components can also contain subcomponents. Anyor all of the components can be contained within or otherwise coupled tothe base. The base optionally forms a housing or a connection point forthe other components that make up the breath analyzer device.

FIG. 1 shows a preferred but illustrative embodiment of a system formeasuring at least one analyte in breath. The system comprises a base inthe form of a base unit (2), dispensing device here in the form of aninsertion mechanism (8) for a cartridge, an optical sensing subsystem(10), a flow facilitator, here specifically in the form of a pneumatichandler (12) and a digitizer (14). The base unit (2) receives breathfrom a user via a breath input (4). The insertion mechanism for acartridge includes means for a cartridge to be inserted, where thecartridge contains a reactive chemistry capable of reacting with atleast one analyte when present in the breath in concentrations of lessthan about 5 ppm to generate an optical change. The optical sensingsubsystem measures an optical change. The pneumatic handler ispreferably included within the base unit, although this is not alwaysthe case. The pneumatic handler allows for the breath to interact withthe reactive chemistry in the cartridge. The digitizer quantifies theoptical change measured by the optical sensing subsystem and outputs adisplay containing information regarding the at least one analyte in thebreath.

The base unit can be any apparatus that receives breath from a user. Incertain embodiments, the base unit contains the pneumatic handler. Inpreferred embodiments, the base unit is portable and capable ofindividual patient use. The base unit may also be capable ofwithstanding (measuring and compensating for) temperature and humiditychanges so as to improve the accuracy of the measurement process.

A breath input can be anything capable of receiving breath from a user,and optionally perform the function of breath metering. The breath inputmay optionally include the step of breath conditioning, but this mayalso be handled by the base unit itself. The breath input can alsoinclude breath sampling, which preferably utilizes a reservoir forcontaining the breath sample.

In general, breath collection involves the collection of breath in theform of a breath sample. Such breath collection may be direct orindirect. For improved relevance of the measurement results made by thebreath analyzer, breath collection can be performed with attention todetails such as: (a) total volume of breath collected; (b) source ofcollected breath (e.g., upper airways vs. alveolar air); (c) number ofbreaths collected; (d) physiological status of the subject prior to andduring breath collection (e.g., rested state with normal breathing vs.active state with increased breath rate vs. hyperventilation, asexamples); and (e) breathing effort of the sample collection mechanism(e.g., does the subject need to breath through a high-resistancecollection apparatus at extended duration, or does the mechanism allowfor normal breath exhalations).

FIG. 2 shows an example of a breath collection bag with integrated flowmeasurement capabilities. A breath sampling bag (20) comprised of wallmaterials impermeable to the analytes of interest and in some cases alsotheir ambient interferents contains a breathing inlet (24) fitted with amouthpiece (22). An upper portion of the assembly houses electronicsand/or mechanical devices useful in analyzing or conditioning breathsamples, including in some cases a visual indicator (26). Theelectronics can consist of a variety of assets, including temperatureprobes, pressure transducers, timing circuits, humidity sensors, andothers depending on the application. Mechanical devices can includeone-way breathing valves, flow restrictors, scrubber or desiccantchambers, computer-controlled or automatic valves, manual valves, andothers. In one embodiment, the one-way valve (24) is designed to matewith a receiver port on a base unit which is equipped with fingers orprotrusions designed to open the one-way valve. This system enables abreath sample to be collected from a user and to be contained within thesampling bag without user interaction. Attaching the bag to the baseunit allows the fingers or protrusions to open the one-way valve (forexample, a flapper valve) so that the contents of the bag can be removedby, for example, a pneumatic handler of the base unit. No manualinteraction with the valve is required by the user. Also shown in FIG. 2is a user interface button (28), exemplifying a possible interaction ofthe user with the electronics, such as to start a timer. A second end ofthe bag (25) can be fitted with similar facilities. For example, fittingthe lower portion (25) with a second one-way valve, such that the userbreathes into a first one-way valve (24) and out through the second (25)results in the last exhaled portion of air being captured in the bag.This can be used to sample, for example, the deep alveolar airspacewhereas without the second one-way valve the air collected is the firstportion blown into the device. The bag may likewise be fitted at otherpoints, for example on the sides or front/back faces.

It is often desirable to condition the breath prior to or as part of theanalysis. Particular examples of breath conditioning include: (a)desiccation (e.g., removal of water); (b) scrubbing (e.g., removal ofcarbon dioxide or volatile organic compounds); and (c) heating orcooling of the gas stream (condensation prevention/instigation). Thebreath condition function, if performed, can be carried out by thebreath input or a separate system.

The optical sensing subsystem can be any detector or other sensor thatis capable of measuring an optical change. This may be a directmeasurement of optical change. It may also be an indirect measurement ofoptical change (e.g., transduction through other energy states). Theoptical change may involve any of the following, alone or incombination, without limitation: reflectance, absorbance, fluorescence,chemiluminescence, bioluminescence, polarization changes, phase changes,divergences, scattering properties, evanescent wave and surface plasmonresonance approaches, or any other optical change known to those skilledin the art.

The optical sensing subsystem may be contained within the base unit orit may be a separate module that is plugged into the base unit. Theoptical sensing subsystem may be single use or it may be used multipletimes. The optical sensing subsystem may also comprise an array ofdetectors that work in tandem to measure the optical change.

In system designs utilizing any of reflectance, absorbance andfluorescence, excitation light is supplied to the system and changes inthat light are tracked in relation to changes in the chemical state ofthe sensor system. It is preferred to minimize the amount of unmodulatedlight that enters the sensing subsystem and to measure only the lightthat is being changed by the chemical system. For example, a chemicalsystem that produces a maximum absorbance change at 400 nm is preferablyimplemented with excitation light at 400 nm as opposed to unfilteredbroadband light sources such as incandescent lamps. However, if a baseunit is intended to measure numerous chemistries with various spectralcharacteristics, broadband excitation sources may be preferable.

Excitation sources include, but are not limited to, incandescent lamps,such as tungsten filaments and halogen lamps; arc-lamps, such as xenon,sodium, mercury; light-emitting diodes, and lasers. Excitation light maybenefit from conditioning efforts, such as filtering, polarization, orany of the other methods known by those skilled in the art. For example,allowing only light of the wavelength that matches the wavelength of thechemical system's peak response is useful in increasing the signal tonoise ratio of the optical system.

Each of these modalities can be employed with spot interrogations aswell as with scanning mechanisms, either one or two-dimensional. Ascanning system can be useful in breath measurement devices, especiallywhere analyte concentration varies along an axis and where thatvariation is indicative of analyte concentration in the breath.

FIG. 4 and FIG. 5 depict optical configuration embodiments useful forendogenous breath sensing. FIG. 4 depicts a general layout for anoptical sensing subsystem configuration comprising a camera (36) inrelation to a light source (38) and cartridge (40). FIG. 5 depictssimilar components from a top-view, illustrating the relative angle ofthe excitation source (42) to the incident plane of the cartridge (44)and to the focal plane of the camera (46). Such an embodiment reducesglare from the excitation source and is suitable for capturinghigh-quality images of the sensor chemistry. The images can be processedto derive or to interpolate from correlations of analyte breathconcentrations and developed color. A camera is especially well-suitedto base systems where multiple chemistries are to be detected due to theadditional power afforded by both a wide spectral range, a degree ofspectral sensitivity (images are captured onto red, green, and bluepixels), and a high degree of spatial resolution. In particular, spatialresolution allows very simple instrumentation setups to be used for awide range of applications, for example quality assurance. Otherembodiments such as semiconductor photodetectors can provide lowprocessor overhead and compact size.

In accordance with an aspect of the present invention, a cartridge isprovided for use in carrying out various methods according to theinvention. The cartridge optionally but preferably forms a separatecomponent from the remainder of the breath analysis device, andcomprises reactants or consumables used by the various tests oranalyses. Again, optionally but preferably, the cartridge is configuredso that, when disposed in its “inserted” position in the breath analysisdevice, it forms a substantially light-tight seal so that ambient lightor like radiation that may adversely impact features of the methods suchas the colorimetric analyses are avoided.

Cartridges according to presently preferred embodiments in the contextof the present inventions include at least one “packed bed” reactionzone or cavity, which reaction zone or cavity may contain the one ormore reactants as described herein below. Such cartridges also maycomprise breath conditioning means, such as a flow controller orpressure regulator, a desiccant (e.g., calcium chloride), a temperaturecontroller, and the like. The cartridge also may comprise a cavity orportion for containing a “developer solution” or like fluid tofacilitate the analysis, e.g., as described more fully herein below. Insome preferred embodiments, each cavity is separated from the others bya porous retention medium. One example is a porous polyethylene disk.

Preferred cartridges comprise reactive chemistry capable of reactingwith at least one breath analyte, and preferably at least one endogenousbreath analyte. There are a variety of cartridge configurations that canwork with systems according to the invention for measuring at least oneanalyte, preferably an endogenous analyte, in breath.

In one embodiment, cartridges comprise an encasement that has a flowpath for breath that is further coupled to an automated reactioninitiator that allows the developer solution to contact the reactivechemistry. Cartridges preferably contain a porous media located adjacentto the reactive chemistry. The cartridge may contain a single reactivechemistry or a plurality of reactive chemistries.

In another embodiment, cartridges contain a pneumatic loader thattransports developer solution through the cartridge.

In yet another embodiment and aspect of the invention, cartridges blockambient light when inserted into the base unit and preferably comprise ahandle. As noted herein above, where internal system components such asthe interactants, intermediate products, etc. are light-sensitive, thebase may comprise an exterior surface that forms an interior and shieldsthe interior from ambient light, wherein the exterior surface comprisesan aperture; and the cartridge may comprises a shroud that substantiallyconforms to the aperture to shield ambient light from entering theaperture when the cartridge is coupled to the base.

Cartridges can be designed into various shapes and sizes to facilitatedifferent applications. In one embodiment, the cartridge is comprisedof: (a) reactive chemistry, (b) a first chamber containing a firstdeveloper, and (c) a second chamber containing a second developer. Thefirst and second developer can be the same or different. In anotherembodiment, the cartridge is comprised of: (a) reactive chemistry, (b) achamber containing a developer, and either (c) mechanism for couplingthe cartridge to a pneumatic loader or remover, or (d) mechanism forcoupling to a reaction initiator. In a preferred embodiment, thecartridge requires no external liquid flow to the cartridge.

An exemplary general schematic of cartridge design is shown in FIG. 13.This cartridge is preferably used for optical detection, and preferablyincludes reactive chemistry that can be used to detect endogenouslyproduced analytes in human breath. Here, the reactive chemistry (128) iscontained within a cartridge housing (120) consisting of a single piece.Preferably, but not necessarily, the housing is comprised of materialthat is optically clear. There is a membrane (122) that separates thereactive chemistry from a breath conditioner (124). In this embodiment,the breath conditioner (124) is a desiccant, but this may also be ascrubber or pre-concentrator. The breath conditioner is kept tightlypacked by a porous membrane (126). In some embodiments, a peelable orpiercable barrier material can be affixed to the underside of thecartridge to enhance storage of the reactive chemistries and breathconditioners. On the other side of the reactive chemistry is a retainer(130). The retainer serves to keep the reactive chemistry tightlypacked. This retainer can be molded compression fittings, on-cartridgegaskets, o-rings, etc. Atop this retainer is a porous media (132). Theporous media is designed to allow liquid developer solution (133) toflow towards the reactive chemistry. In an alternative embodiment,components (130) and (132) are replaced by a single component that canbe both compressive fit into the packing pocket and porous. Hydrophilic,porous polyethylene disks are useful for this purpose. Developersolution is contained within a breakable ampoule (133) that sits withina receptacle in the upper portion of the cartridge housing (131), whichis formed with vertical channels to facilitate venting of air whendeveloper solution flows down into the channel filled with reactivechemistries (128). The ampoule-containing receptacle (131) is sealedwith a piercable membrane (134). Once the cartridge is inserted in thebase unit, the piercable membrane and the piercable container arepierced by the reaction initiator of the base unit so that liquid flowsto the reactive chemistry. To ensure that residual liquid does not leakout post-use of the cartridge, there is a rubber septum (136) that sealsthe cartridge. The cartridge preferably is designed such that thedeveloper solution is “absorbed” by the reactive chemistry and/orconditioner (e.g., desiccant) such that it does not leak through thebottom of the cartridge. One optional addition is coupling to apneumatic loader or remover (not shown). This pneumatic loader/removeracts as a pump and pulls/pushes the developer solution through thecartridge. Thus, while the cartridge can be oriented such that theliquid interacts with the reactive chemistry due to gravitational pullor wicking, it can also be designed to allow for automated, activeinteraction via a pneumatic loader/remover.

FIG. 14 shows one alternative to the retainer (130) of FIG. 13 forcontaining reactive particles (128). A plastic cartridge (138) forms themain housing for a packed bed of reactive materials (142). A permeableretainer (140) is affixed on the underside of the column as discussedelsewhere. A porous material, for example plastic, metal, ceramic; orfibers such as glass or metal wool is compression fit into the channel.The porous plug is pressed tightly against the packed materials (142) toprevent shifting during usage or transportation.

The porous seal (130) exemplified in FIG. 13 is preferably comprised ofa material with the following properties: fine pore (able to retainsmall particles, for example 75 micron particles), high open area (lowpressure drop, low resistance to flow), inert to analyte of interest,amenable to pick and place automation, able to adhere sufficiently tothe substrate. Materials in sheet form are often amenable to massproduction. Sheets of various substrates are easily pressed intolaminates. A sheet that is porous to begin with is easily processed intoretention disks. FIG. 12 displays an example of a substrate sheet thatcan be pressed into retention disks. A sheet of thin polyimide(0.001″-0.003″) with adhesive backing is punched with an array of holes(110) (for example Devinall SP200 Polyimide film with FastelFilm 15066adhesive backing). A sheet of fine woven nylon mesh (307×307 mesh,9318T48 from McMaster-Carr) is pressed into a laminate with the punchedpolyimide. The laminate is then punched with a larger diameter tool tocreate laminated disks with a porous center (118). The outer regioncontains a topside annulus of polyimide. Such disks are easily picked upby vacuum means to be positioned easily, even into deep recesses. Disksare adhered to receiving surfaces using heat pressing tools. Theparticular adhesive melts at 66 C, well below the melting points ofnumerous plastics suitable as cartridge wall materials. Disks can befashioned by this method using commercial rotary cutters and othercommon production tools. These disks are especially well-suited toretaining reactive media in deep wells, for example (324) in FIG. 26,discussed infra.

Foils and numerous other plastics are also available with adhesivebacking. Polyimide top layers can be preferable to foil layers in someattachment methods since foil layers can have a greater tendency toseparate from their adhesive backing during certain heat pressingprocesses, especially where the contact surface area is large. Polyimidemay be preferable to other plastics due to its potentially high heattransfer and resistance to heat damage, especially when thermal gradepolyimides are used.

Liquids can be contained in the pockets of cartridges, using thecartridge material as side walls with foil or other membrane barriersadhered to the cartridge surfaces. For aggressive solvents, for exampledimethylsulfoxide or methanol, such solutions may be temporary due tosolvent attack of the adhesives. One embodiment of the present inventionuses a separate part to contain the liquid reagent. This allows completematerials control of liquid contact (the walls of the cartridge do notneed to be a materials concern as far as solvent interaction isconcerned). Various “cans” of liquid can be configured, and these canscan be dropped into an open pocket in the top piece of a cartridge.Preferably a liquid can or ampoule is completely inert to the retainedliquid. FIG. 15 shows four embodiments of a piercable foil ampoule,described in the following paragraphs.

Breakable solvent ampoules can be manufactured by a variety of methods.For example, in one case, a flanged conical foil base (152) is welded orotherwise adhered to a weldable or heat-sealable intermediate material(150) to form the bottom half of a clamshell. A top foil layer (146) islikewise attached to a weldable or heat-sealable intermediate material(148) to form the top half of the clamshell. The bottom half is thenfilled with volatile liquids and the top half ultrasonically welded orheat sealed to the bottom half. The volatile liquid is contained withinup to four barriers: the foil material forming the major contactsurface, the weldable/sealable intermediate material (for example lowthermal conductivity thermoplastic), the weld joint between the foil andthe plastic (adhesive), the weld joint between the weldable intermediatematerials (low thermal conductivity thermoplastic). This configurationis useful because (a) it allows an adhesive time to cure independent ofsolvent presence (the adhesives can be fully cured before filling of thesolvent), thus enabling a wide range of adhesives to be employed; (b)conductive heating caused by ultrasonic welding is shielded by lowthermal conductivity thermoplastic, eliminating or controlling theamount of fill solvent lost to evaporation during ultrasonic welding.

A thermal barrier material is another example of a breakable solventampoule. A second case ultrasonically welds the two foil half clamshellsto one another, using a bottom half insert material as a thermalbarrier. That is, a top foil (154) is attached to the bottom foil (156)by direct ultrasonic welding of the metal foil. The solvent ispre-loaded for welding, thermally protected by a thermal barrier, suchas a hollowed out wax cone (164). The thermal barrier must protect thesolvent from conductive heating caused during ultrasonic welding, but itmust also be easily pierced. Other materials, such as thin plastics,rubber, or spray-on silicone adhesives may also be suitable.

An adaptation of the thermal barrier method is to perform ultrasonicwelding in the presence of appropriate heat sinking. The ultrasonic weldjig contains an annular clamp made of highly conductive metal. The clampengages the top and bottom metal foils inward from the outer locationsof ultrasonic welding such that any heat conducting away from the weldjoint sinks into the conductive clamp. Alternative methods of heatsinking, such as blowing the bottom foil with cold air may also besuitable, depending on the solvent in use.

A third method for solvent encapsulation relies on a crimp seal betweena top foil (158) and a bottom foil (162). A wax gasket or gasketcomprised of solvent-resistant material (160) is included between thelayers to increase the retention time of the volatile liquid into theampoule. The gasket material must be chosen with the appropriateresilience and barrier properties to the solvent of interest.

Ampoules can also be blow-molded from numerous materials includingglasses and plastics. These single-material ampoules are constructed ofthin walls to enable ampoule piercing, but sufficiently thick walls toobtain the necessary barrier properties.

Metals are excellent as barrier materials and can be sealed in gas-tightfashion through crimping (such as a beverage can). Miniature ampoulesmade of aluminum and other metals can be manufactured and dropped intothe head portions of disposable cartridges.

FIG. 16 shows certain embodiments of a piercable ampoule. In thisembodiment, a cold-formed foil (176), or other formed, piercable barriermaterial, is attached into the head portion of a base plastic carrier(172) using points of adhesive. These points may make contact with aseries of bosses (188) and are intended to adhere the floor of theampoule to the base plastic carrier in a non-airtight fashion. The floorof the ampoule (176) is filled with solution, and a temporary barrier(180) may be affixed to seal the liquid. The temporary barrier can beaffixed through pressure sensitive adhesives, thermally set adhesives,or any other convenient method. The adhesive for the temporary barrierdoes not need to resist and retain the solution beyond the time requiredto complete the sealing process. A circular bead of adhesive (182) isnext applied. This adhesive forms a permanent barrier for the entrappedsolution, but a temporary barrier (180) allows the permanent barriermaterial (182) to cure independent of solution activity. The liquid iscapped with a disc of barrier material (184). A separate material (186),such as a rubber septum, is optionally placed to prevent temporarypassage of liquid after the barriers have been broken.

This method can be used to retain particles in a packed state. That is,by positioning of a compressible, porous material (190) directly beneaththe bottom floor (176), particles can be immobilized.

FIG. 17 shows embodiments of a piercable can. In this example, athin-bottomed can (192) is cast of a thermoplastic material. Afterfilling with the desired liquid, a thin barrier material (a laminatedfoil with a thermoplastic layer, for example) can be attached via anappropriate method, such as ultrasonic welding or heat-sealing. Asnecessary, more extensive barrier materials (196, 198) can be affixedafter the can is filled with liquid. Optionally, depending on thematerial requirements of the liquid to be contained, barrier materials(196, 198) can be attached directly to the can through pressuresensitive adhesives, thermally set adhesives, or other methods (notethat the can does not need to be constructed of thermoplasticmaterials). A variation on this design uses a thick-walled plasticcylinder as the body of the ampoule and is sealed on both ends withpiercable barrier materials.

Single analyte cartridges can be configured in numerous ways tofacilitate various chemical reactions. Sequential columns of dryreagents can be packed into stacked columns (where shifting of particlesis not a concern) or into partitioned pockets within the device. Someexamples are shown in FIG. 18 and FIG. 19.

In FIG. 18, three distinct dry reagents (200, 202, 204) are packed intoa single column. Porous membranes (206) and (208) are in place to retainthe reagents. Reagents can be of dissimilar size when membranes are inplace. Additional reagents can be packed using increasing diametersections, such that flat ledges are created whereupon retention meanscan be affixed.

In FIG. 19, reagent stacking is shown. When distinct reagents of similarsize (212 and 214) need to be immobilized, they can be packed into asingle column as shown. Larger particulates (218) will need means ofseparation and retention (216). One method of separation makes use ofthin disks of porous material, such as nylon mesh as described in FIG.12, but porous plastics or other porous media can be used in additionalembodiments. The outer ends can be sealed using retention membranes(210) and (220). It is often desirable to pack columns with reagents insuch a manner that the reagents are not free to move. In this case,materials can be held using compressible, porous media. FIG. 20illustrates such a configuration. In this illustration, a cartridge iscomprised of two pieces, a top (222) and a base (228). A first dryreagent (232) is packed into the lowermost pocket of the base, retainedby two porous membranes (230 and 234). A second dry reagent (226) ispacked into the central column of the cartridge. At the topmost end ofthe central column, a wider bore has been molded to accommodate slightoverfilling of the dry reagent (to relax filling tolerances) and tofacilitate compression of the reagents with a porous, compressiblematerial. This material, when compressed by the top (222), still allowsfluidic communication through the top and bottom pieces whilecompressing the dry reagents 226) to keep them immobile.

Liquid reagents can be packed into sensor cartridges to facilitatenumerous chemical reactions useful in breath analysis. FIG. 21 shows anexample of how a liquid reagent can be immobilized onto a cartridge andhow it can be released at the time of reaction. In a top piece of acartridge (240), a containment means (238) is provided for the liquidreagent. This can be a distinct component (238) that is dropped into apocket in the top piece (240) or it can be integral to the top piece. Inany case, this reagent ampoule (238) can contain liquid reagent betweentwo piercable membranes (252) that are impermeable or otherwise amenableto the reagent of interest. A needle (236), solid or hollow, is pressedthrough the membranes at the required time, causing the liquid reagentto flow through a conical cutout (250) in the cartridge and through adowncoming channel (246) toward the reactive bed (244). In thisconfiguration, the seal between the top piece (240) and base piece (242)is not airtight (to allow gas flow from the bottom of the reactive bed(244) through to the top and out the sides). Thus, the liquid reagent ispreferably of low viscosity and appropriate surface tension such thatthe liquid drops all the way to the top of the reactive bed and is drawninto the reactive bed when a suction pump (248) is activated.

FIG. 22 provides another embodiment. In this alternate configuration, ahole (260) is cut into the top piece so as to provide a gas exit portwhen the top piece (254) and the bottom piece (256) are fastened with anairtight seal. In this case, gas is flown over the reactive bed and outthe exit port (260). Next, a pin (262) is pressed through a top and thenbottom barrier to free the contained liquid and to create a hole toallow gas to fill the vacated space. The liquid fills a downcomingchannel (264), blocking the exit port and creating a liquid seal so thata suction pump (268) can pull the liquid through the channel and throughthe reactive bed.

An extension of the liquid containment/release mechanism as describedabove allows multiple liquid reagents to be integrated into a singlecartridge. FIG. 23 and FIG. 24 illustrate examples of a multi-liquidcartridge. In FIG. 23, two reagent wells A and B contain two reagents(or one reagent, if desired) between breakable seals as discussed. Thedowncoming channels are merged into a single line. When the first sealis broken, liquid from A fills the downcoming channel as before, whereit is then suctioned away by a connected pump. Next, the sealscontaining liquid B are broken, and the same procedure is followed. FIG.24 shows a top piece that contains four such containers of liquid. Thismethod allows very sophisticated fluidic handling to be done withreagents that are located on a single disposable piece.

Although chemical reagents may be consumed with each reaction,cartridges of the present invention need not be limited to single-use.Multiple use devices can be comprised of strips or carousel wheels ofdevices in a single substrate. This same form factor can be used toallow multiple analytes to be measured in a single breath sample, eitherwith sequential or parallel processing.

FIG. 25 shows some cartridge designs to enable these applications.Displayed on the left side of the diagram is a strip or blister pack ofreactive channels. Each of the four channels (292, 294, 296, 298)depicted can be filled with identical or different reagents, dependingon whether the application is to measure, as examples, acetone on fouroccasions, acetone and ammonia each on two occasions, or to measure 4separate analytes from a single sample. Each channel can be sealed witha separate foil barrier (300) or with a single foil strip placed overthe entire top portion. Windows to reduce material volume and wallthickness for optical clarity can be fashioned next to each packedcolumn. The base device must contain four fixed channels or moving parts(to move either actuators or the table containing the multi-channelcartridge). Also shown in FIG. 25, multiple channels are incorporatedinto a carousel-type device (306) which rotates to align each channelwith a fixed-position seal breaking/fluid driving head.

FIG. 26 shows an embodiment of a cartridge design that facilitates oraccomplishes the following tasks: (a) sample desiccation, (b) sampleconcentration, (c) sample reaction, (d) built-in fluid direction control(via one non-reversible one-way valve, schematically similar to threeone-way valves), (e) two-phase reagent containment (solid reactivechemistry, liquid developer). (f) inexpensive reagent interfaces(retention means), (g) easy insertion into base device, and (h) lowreagent volume.

The exemplary cartridge in FIG. 26, in connection with appropriatereagents, is appropriate to measure acetone in human breath. Thecartridge is comprised of two pieces that are mechanically fastenedtogether, for example with snap fits. A top piece (312) attaches to abase piece (314). The top piece and base piece, by design, do not forman air-tight seal. Liquid reagent is contained in a pocket (316) in thetop piece. One embodiment consists of a developer solution containedbetween two foil seals, one on the top plane of the pocket and a secondon the bottom plane. Beneath the bottom foil seal, a conical pocket(318) is fashioned to facilitate liquid reagent dropping withoutintermittent air bubble entrapment. Reactive chemistry is packed into acolumn (322) running through the center of the base piece. To easetolerances on reactive chemistry packing, the top-most portion of thereactive column is widened. A porous, compressible medium is depositedin the top-most, widened column portion such that when the top piece(312) is sealed against the base piece (314), the reactive materialloaded into the column (322) is packed tightly. In general, open cellfoams, both foam-in-place and pre-formed and cut, are well-suited asporous, compressible retention barriers as long as the chemistry iscompatible with the system. Columns that are not packed tightly aresubject to material shifting, a situation which hampers reproducibilityand increases measurement errors. Desiccant materials are packed into alower, wider column (326). A porous seal (324) is attached to theceiling of (326) to provide a gas-permissive retention mechanism for thereactive material. In one embodiment, woven nylon mesh provides thismeans while incurring negligible resistance to gas flow. A similarbarrier (330) forms the floor of (326). The base of the cartridge isformed to facilitate compression against a trapped gasket in the basedevice to enable leak-free communication with the gas delivery plumbing.Pockets have been fashioned into the cartridge walls to enhancecolorimetric detection. The pocket depth is selected to minimize wallthickness while simultaneously preserving the mechanical integrity ofthe cartridge, especially in relation to the wider bores required forthe pockets that contain accessory reagents. The wall angle, withrespect to the four relatively square sides of the cartridge, can beadjusted to promote effective illumination and to attenuate harshreflections of excitation light in particular.

One preferred example of how a cartridge interacts with a base unit isin the following manner. First, the user opens a door through the wallof the base device and places the cartridge into a cartridge receptacle.No significant force is required of the user to make the insertion, andinsertion orientation is restricted by mechanical stops. Either of two(of the four) sides of the cartridge must be oriented toward the opticalsetup. A cartridge receptacle that receives the cartridge at an angle(whereby the top portion of the cartridge is inclined away from the userwith respect to the bottom portion) increases user accessibility andcomfort during cartridge insertion. Once the cartridge is loosely placedwithin the device, mechanical means are provided whereby the topside ofthe cartridge is compressed against a captive gasket in the base device.See FIG. 9 and FIG. 10. This compression forms a face seal between thegasket and the bottom of the cartridge, providing a leak-free fluidicconnection capable of withstanding the driving pressure required to movebreath samples and developer solution through the cartridge and itsvarious compartments. Once the cartridge is in position, a breath sampleis collected through various means, for example a breath collection bagor sidestream sampling. Once a sample of gas is ready for measurement, apneumatic handler is actuated which withdraws breath gas from the gascollection vessel and pumps it first through the desiccant bed, nextthrough the reactive column, and out through the cartridge. See FIG. 27.The cartridge is designed to be open to gas flow at both ends. Thebottom side (desiccant side) is open through a woven mesh barrier, thetop-side is open through the non air-tight sealing of the top piece(338) to the base (340). Thus, when gases are pushed through the bottomof the column, they can vent through the top although the developercontainment barriers have not been broken. After the proper volume ofbreath sample has been pushed through the column at the selected rate offlow, the developer solvent containment means is ruptured. See FIG. 21.A sharp pin (236) is driven through the lid of the cartridge such thatit breaks the top barrier (252) of the containment means first, then thebottom. Slower pin drive speeds and appropriate contained volumes ofdeveloper are preferred to prevent developer spillage during rupture.Also preferred is the ability of the containment means to withstanddeformation during rupture when such deformations result in spilleddeveloper solution. Once the developer is released, it fills the conicalpocket (250). The conical pocket assists in creating a liquid seal(251), such that when fluid is pulled through the column (246) there isa continuous pull of developer into the column. The amount of developerpulled through the column can be controlled (open-loop) by adjusting theduration of the pulling pump's on cycle, or closed-loop means can beemployed. An imaging system (see FIG. 4 and FIG. 5) is used to recordcolorimetric responses which result from analyte reaction with thereactive bed and developer solution. Developer solution can be largelycontained in the desiccant bed. Optional top and bottom septa can bebuilt into the cartridge when potential user exposure to especiallydeleterious solvents should be prevented.

FIG. 13 shows a preferred method for single-analyte cartridgeconstruction. A single piece of molded clear plastic (120) such asacrylic forms the cartridge housing. A particle retention barrier (122),as previously described, is attached to the bottom of the flow channelbut is comprised preferentially of thermal adhesive-backed (Fastel15066, 3 mil thick) polyimide (Devinall, 2 mil thickness) with wovennylon center (198×198 mesh, 0.0031″ opening, 49% open). Desiccantmaterial (30-60 mesh anhydrous calcium chloride) fills a desiccantchamber (124). A particle retention barrier (126) similar to (122) isplaced on the bottom to contain a desiccant. The reactive materials(100-140 mesh aminated and nitroprusside-attached particles in a 2:1ratio) are placed in the flow column (128), and the top portion of thechannel opens to facilitate low-tolerance filling. A porous material(130) such as glass wool, stainless steel mesh, or porous hydrophilicpolyethylene plastic (preferentially) is placed over the reactiveparticles. In some embodiments, the reactive particles (128) and porousbarrier (130) may need additional means to be compressed tightly againstthe particles. An o-ring, external toothed push-on ring, or deformableretainer ring may be suitable for this purpose, but porous plastic canmake its own compression fit without the need of these means. Apiercable liquid ampoule (133), comprised preferentially of athermoplastic, heat-sealed can with pierceable barriers on top andbottom, is placed into a holding housing in a manner that does notocclude airflow. The top of the cartridge is sealed with a piercablefoil (134) and a liquid barrier septum layer (136), such that liquidcannot leak through the lid after the cartridge has been used.

FIG. 28 shows a preferred method for using the cartridge discussed inFIG. 13. With the piercing needle (342) in the fully retracted position(A), the top barriers (344) have not been breached and airflow throughthe cartridge is not possible. With the needle in a first extendedposition (B), the top barriers are breached such that gas can flow fromthe bottom of the cartridge through the various porous barriers,reactive bed, around the liquid ampoule, and through the hole in thepiercing needle (348). In a second extended position (C), liquid isreleased from the ampoule (3) and is pulled by suction force of a pumpor by wicking downward through the reactive bed. A needle in the baseunit (343) can be used to pierce a bottom barrier material to allow gasflow into the cartridge. This method allows the cartridge to be sealedfor storage and shipping and to be automatically pierced upon usagewithout extra user steps. Also, the septum on top and extra barrier onbottom can be used to contain the liquid inside the cartridge after use.Note that the barrier to contain desiccant or other conditioningmaterials is not shown in this figure.

In accordance with one aspect of the invention, a method is provided foranalyzing acetone in breath. The method comprises providing a cavity,locating within the cavity a primary amine disposed on a first surface,locating within the cavity a nitroprusside on a second surface distinctfrom the first surface, wherein the nitroprusside is coupled to thesecond surface using a coupling agent comprising an anion exchange resinin an acidic environment, wherein the acid environment comprises an acidhaving a vapor pressure of less than about 1 atm at 22 C in its 99% pureform, the primary amine on the first surface and the nitroprusside onthe second surface comprising cavity contents having a reference opticalproperty, causing the breath to move into the cavity so that it contactsthe primary amine and the nitroprusside to cause or facilitate a changein an optical property of the cavity contents relative to the referenceoptical property, and analyzing the breath for the presence of theacetone using the change in the optical property.

A summary of a preferred implementation of this method is shown in FIG.31. This method can be advantageously implemented using the breathanalysis device shown in FIG. 1 and described herein above, or in the'862 Application. The cavity according to this method preferablycomprises an enclosed, and preferably elongated, volume or zone in whichone or more reactants (most notably the primary amine) can be containedand wherein reaction of the acetone with the primary amine and/or otherreactants (e.g., the nitroprusside) can take place. In presentlypreferred implementations of this method, the cavity comprises theprincipal cavity or chamber in the cartridge as shown in the drawingfigures.

Moisture may have deleterious effects on the constituents and reactionsinvolved in this method, as more fully described herein below, and thusthe cartridge also preferably comprises a moisture control component toreduce or substantially eliminate moisture from the breath sample as itenters the cartridge, and before the breath sample is passed into thecavity.

As noted herein above, the method comprises locating within the cavity aprimary amine disposed on a first surface. This aspect of the methodpreferably locates one or more primary amines in the cavity or reactionzone in a manner that facilitates contacting of the acetone with theprimary amine or amines and reaction of the two. The primary amine maycomprise any aliphatic primary amine. Aliphatic primary amines arecapable of forming a Schiff base upon reaction with acetone, which formsa color product when coupled to nitroprusside. Primary amines that canbe coupled to a surface are preferable. Examples of such primary aminesinclude amino silanes, such as aminopropyltriethoxysilane. Although notwishing to be limited to a particular theory, the primary amine oramines are believed to react with the ketone body, preferably acetone,to form a Schiff base. The nitroprusside then reacts with the Schiffbase to yield the optical property change.

In presently preferred embodiments and method implementations, theprimary amine comprises an amino silane.

The first surface comprises a surface that will support and immobilizethe primary amine or amines, so that they can be contacted by theacetone in the breath sample and favorably react with the acetone. Thefirst surface preferably comprises a plurality of beads that, when inthe cavity, comprise a packed bed.

In some embodiments, the reactive chemistry is coupled to the surface byusing a coupling agent. “Coupling agents” are broadly defined aschemicals, molecules or substances that are capable of coupling (seedefinition for “react”) a desired chemical functionality to a surface.Preferred coupling agents either have branched chemical functionalitiesor are capable of branching during coupling with the surface. “Branchedchemical functionalities” or “branching” refers to having more than onechemically reactive moiety per binding site to the surface. Branchingmay be contained within a single coupling agent or may be achievedthrough the reaction of several coupling agents with each other. Forexample, tetraethyl orthosilicate may be mixed with aminopropyltrimethoxysilane for enhanced branching during the reaction.

There are numerous coupling agents known to those skilled in the art. Inthe class of silanes, there are literally thousands of functionalchemistries attached to a silane. Silanes can be coupled to dozens ofsurfaces, with a preference for silica surfaces and metal oxides, andare capable of de novo surface formation. Examples of common functionalsilanes include aminopropyl trimethoxysilane, glydoxypropyltriethoxysilane, diethylaminopropyl trimethoxysilane and numerousothers.

Coupling agents possessing a free amine are readily coupled to surfaceswith epoxides, aldehydes and ketones, among other chemical moieties.Coupling agents with epoxides, aldehydes and ketones can also be usedwith surfaces containing a moderate to strong nucleophile, such asamines, thiols, hydroxyl groups and many others. Some coupling agentsare attached to the surface through a free radical reaction, such asacrylates and methacrylates among others.

Some coupling agents do not directly react with the breath analyte.Rather, they are intermediate agents. An “intermediate agent” is acoupling agent whose chemical functionality is to react with yet anothercoupling agent. For example, diethylaminopropyl trimethoxysilane is anintermediate agent in the reaction with acetone. It does not directlyreact with acetone, but reacts with sodium nitroprusside, which in turnreacts with acetone. Another example of an intermediate agent would bethe use of glycidoxypropyl triethoxysilane, whose epoxide functionalgroup could be reacted with a host of other molecules to achieve adesired functionality. Numerous intermediate agents are known to thoseskilled in the art.

In presently preferred embodiments and methods, the beads may becomprised of silica, quartz, aluminum oxide, alumino-silicates, silicon,copper, tin oxide, talc, inorganic oxides, or combinations thereof. Apreferred embodiment comprises silica. The optimal size of the silicagel varies with the flow rate and desired detection limits. In general,the smaller the silica gel particles, the larger the surface area forextraction of acetone. With smaller silica gel particles, a higherextraction efficiency is achieved, higher flow rates can be used andless reactive material is required. In some embodiments, a short,intense color change is desired. In some embodiments, silica gel size isbetween about 20 and about 270 mesh, between about 100 and about 200mesh, between about 130 and about 140 mesh.

Preferred coupling agents for coupling the primary amine or amines withthe bead surface comprise aminopropyltriethoxysilane (APTES) anddiethylaminopropyltrimethoxysilane (DEAPMOS).

The deposition of primary amines onto a surface can be accomplished in anumber of ways. Most any non-volatile primary amine can be readilydeposited onto a variety of surfaces by placing the amine in contactwith the surface and drying. Drying can be done under vacuum or atelevated temperatures. Mixing while drying helps to ensure evendeposition.

Some amines such as amino silanes, can be covalently attached to thesurface, which is useful in preventing elution of the amines duringsubsequent solvent based reactions. In practice, the inventors hereofhave used Tris amine and/or aminopropyltriethoxysilane (APTES) coupledto silica gel with equal effectiveness. The latter covalently binds tothe silica gel and is not washed off during solvent based reactions.

“Nitroprusside” is an anion with molecular formula [Fe(CN)5NO]2-. Theuse of the term herein also includes its various salts, such as calciumnitroprusside or sodium nitroprusside. Nitroprusside is also referred toin the literature by different names, e.g., nitroferricyanide.

When used as a reactant or reagent, it is typically in the form of asalt, a common example of which is sodium nitroprusside (“SNP”). Thenitroprusside according to presently preferred embodiments and methodimplementations comprises, and more preferably consists of or consistsessentially of, sodium nitroprusside, although it is possible to useother salts or constituents that comprise the nitroprusside ion.

In accordance with this aspect of the invention, the second surfacecomprises one or more surfaces that support the nitroprusside. Itpreferably comprises a plurality of beads, which may but need not havethe same composition or structure as the first surface supporting theprimary amine. In presently preferred embodiments and methods, thesecond surface comprises a plurality of beads comprising silica, quartz,aluminum oxide, alumino-silicates, silicon, copper, tin oxide, talc,inorganic oxides, or combinations thereof.

The nitroprusside is coupled to the second surface using a couplingagent, preferably comprising an anion exchange resin or a tertiaryamine. The coupling agent comprises anionic exchange resins per se, aswell as polymeric materials that have anionic exchange properties orfunction a priori. Diethylaminopropyltrimethoxysilane is a presentlypreferred coupling agent.

In this method, the coupling the nitroprusside to the second surface iscarried out in an acidic environment wherein the acid environmentcomprises an acid having a vapor pressure of less than about 1 atm at 22C in its 99% pure form.

An acid other than a hydrogen halide is preferred. Hydrogen halidesinclude the group of hydrogen chloride, hydrogen iodide, hydrogenbromide, and hydrogen fluoride. Hydrogen halides are gases in their pureform. Hydrogen halides mix with water to form acids. Hydrochloric acidis a mixture of hydrogen chloride gas and water. Pure hydrogen chloridehas a vapor pressure of 43 atm at 22 C and a boiling point of −85 C.Hence, hydrogen chloride is an extremely volatile gas that can besomewhat stabilized in the presence of an excess of water. Since wateris inhibitory to the reaction with acetone and must be removed, theremaining hydrogen chloride is volatile. It diffuses freely over time tothe primary amines, lowering the reactivity of the primary amines.Further, without the hydrogen chloride to quench the tertiary amine, thetertiary amine is able to attack and degrade the nitroprusside. Saltsthat contain halide anions should also be avoided to prevent loss ofprotons through the formation of gaseous hydrogen halides.

Preferred acids include acids with a pKa less than about 4 and a vaporpressure less than about 1 atm at 22 C in its 99% pure form. Preferredexamples include sulfuric acid, oxalic acid, nitric acid, phosphoricacid, perchloric acid, but there are many others known to those skilledin the art. A preferred acid for this purpose is or comprises sulfuricacid. Acids with a pKa less than 4 are capable of lowering the pH of thetertiary amine below 4. With a vapor pressure of less than 1 atm at 22 Cin their 99% pure form, they are relatively stable over time. Thisreduces the migration of protons from the tertiary amine to the primaryamine and better preserves the nitroprusside and reactivity of theprimary amine.

The primary amine on the first surface and the nitroprusside on thesecond surface comprise the principal contents of the cavity inpresently preferred embodiments and methods. These contents collectivelyhave certain optical properties, such as color, color variation,transparency or translucence, optical absorptivity, and the like. Whenviewed collectively, e.g., when viewed macroscopically with the unaidedhuman eye or with a standard digital camera, have a general color. Inthe initial state of the analysis, with the reactants (the primary amineand nitroprusside) intact, the cavity contents have an initial orreference optical property or set of properties. As reactions occur asdescribed herein, the optical property or properties are expected tochange as a result of those reactions. These changes in the opticalproperty or properties can then be sensed and measured, and thus can beused to assess the extent of the reaction, and the nature and amount ofinitial reactants, most notably the presence and amount of acetone inthe breath sample.

The reference optical property preferably comprises a reference color,and the change in optical property preferably comprises a change incolor with respect to the reference color.

The cavity has a linear dimension, and the reference optical propertycomprises a reference distance of the reference color along the lineardimension. The change in optical property preferably comprises ameasured distance along the linear dimension of the change in color withrespect to the reference color.

As the breath sample is moved into the cavity, it contacts the primaryamine and the nitroprusside to cause or facilitate a change in anoptical property of the cavity contents relative to the referenceoptical property.

A number of means may be used to react the primary amine or amines withthe ketone body, and this is not necessarily limiting. The contactingand reaction may take place, for example, in solution.

Although not wishing to be bound by any particular theory or mechanismof reaction, it is believed that acetone optimally forms a Schiff basewith a primary amine under slightly acidic conditions, e.g., at about apH of 5. In practice, however, the inventors hereof have not observed asignificant difference in extraction efficiency or kinetics of reactionwith primary amines (Tris, APTES, etc.) on silica gel from a pH of 1.0to 11.0. The rate limiting step is believed to be the reaction of theSchiff base with nitroprusside. Binding APTES to silica gel with no acidadded has a pH of roughly 10.

The presence of water in the sample or in the substrate where theprimary amines are found reduces the binding efficiency, presumablythrough the reversal of Schiff base formation. In practice, theinventors hereof have discovered that humid samples cause the acetone tobind over a longer distance and slow the color development. Extractionor reduction of the water from humid samples to an acceptable low levelis a requirement to maximize acetone extraction efficiency and kineticsof color formation.

Optionally but preferably, the method comprises using a developersolution to facilitate the change in the optical property. In thepreferred embodiments and method implementations, the developer solutioncomprises 0.5% diethanolamine in 25% dimethylsufoxide, and 75% methanol.The use of the developer solution in this embodiment and in others inthis application serves primarily two purposes: 1) to deliver or enhancedelivery of nitroprusside (i.e., by diffusion or convection) and/or 2)to alter the pH.

As a general principle, the developer solution preferably should beanhydrous and should contain anhydrous components. The presence of waterslows color formation, presumably due to the reversal of Schiff baseformation. It should be noted that reagents that form water (e.g., NaOH)during the reaction may be unsuited for certain applications.

As mentioned previously, SNP can be stabilized in solution through theuse of an acidic medium. Rather than being dried onto a surface, the SNPcan be stored in the developer solution itself. In practice, low pHsolutions (e.g., 1.0) are not ideal for developing the acetone reactionon columns. Presumably, the large number of protons quickly saturate theamines on the surface, creating a large positive charge which extractsthe negatively charged nitroprusside anion from solution before itreaches the reaction site. The nitroprusside is visibly extracted on thesurface in these cases. The low concentration of downstream SNP reducesboth sensitivity and reaction kinetics. However, this obstacle can beovercome by: 1) using a larger amount of developer solution, 2) using asmaller amount of aminated surface, 3) adding the developer solutionfrom the same side that the sample is added, or 4) using a less acidicsolution. A pH of 4 has a much less visible effect, while a pH of 7 doesnot have any observable effect.

Basic developer solutions degrade nitroprusside in solution, althoughhigh concentrations of nitroprusside can be used to quench the base.Generally basic developer solutions are more suitable for reactionswhere nitroprusside is deposited in a more stable acidic stateelsewhere, whether in solution or coupled to a surface.

When coupled to primary amines at a low pH, the pH must be raised abovea threshold level before the Schiff base will detectably react withnitroprusside. The selection of the base to remove the proton isimportant. Only bases with a similar or higher pKa for their conjugateacid will have the strength to rapidly deprotonate the amine. Ingeneral, the stronger the base, the faster and more complete thedeprotonation. Further, concentrations of base in the developer solutionare important for both kinetics and the ability to remove sufficientprotons to raise the pH.

In systems where the developer solution is passed across a relativelylarge amount of protonated amines or surfaces, a pH gradient can form.Where the developer solution first contacts the amines, a high pH willbe created, turning unreacted nitroprusside a yellow color. However,downstream as the base in the developer solution is used up, the pHremains relatively low and the nitroprusside appears a reddish browncolor, which can be confused for a positive acetone signal.

Sufficient developer solution must be passed across the surface in orderto remove the pH gradient and form a constant pH. In practice, theinventors of the present invention have achieved this by either pushingthe developer solution through or by placing sufficient wicking materialon the other side of the aminated surface to pull a large volume ofdeveloper solution through.

When the primary amines are not heavily protonated (i.e., the pH isclose to the pKa of the conjugate acid), the strength of the base can belower. A relatively weak base compared to the primary amine can be usedin this case. This prevents a positive charge from accumulating in thedeveloper solution which can carry nitroprusside downstream.

The method according to this aspect of the invention further comprisesanalyzing the breath for the presence of the acetone using the change inthe optical property. This is preferably achieved by observing andmeasuring the optical property change. In the preferred embodiments andmethods, this is carried out using the digital camera and othercomponents of the optical subsystem, as described herein above and inthe '862 Application.

FIG. 36 shows the sequence of changes in optical characteristics of thereaction zone during the optical detection of acetone in certainembodiments. Panel A shows reactants disposed in a reaction zone priorto delivering the breath sample to the interactant region. Dry reagentsexhibit an optical characteristic at a first reference level. As shownin Panel B, during delivery of the breath sample to the reaction zone,the analyte adheres to the reactant but does not exhibit an appreciableor selective change in the optical characteristic of the reaction zonewith respect to the first reference level. Panel C shows the generationof an optical characteristic at a second reference level caused byaddition of a developer. This state is intended to illustrate theoptical change in the reaction zone due to administration of the liquiddeveloper. In some cases, the second reference level exhibits a changein spectral content from the first reference level, but in other casesthis change may be the result of a refractive index change withoutsignificant spectral shift. In some cases, this state may be so brief asto be unobservable but in other cases there will be a significant dwelltime in this state. Panel D illustrates, for example, the development ofcolor in the reaction zone, comprising an optical characteristic at athird level, which is a change with regards to the second referencelevel. In other embodiments, the change caused by, for example, Panel Dmay be compared to the first reference level of Panel A.

In accordance with another aspect of the invention, a method is providedfor sensing acetone in breath using a breath analysis device. Thismethod, among other things, addresses the instability of thenitroprusside in an aqueous alkaline environment. As noted herein above,a traditional approach to the use of a nitroprusside and a primary amineto detect acetone has been to mix or intermingle the nitroprusside andthe primary amine prior to introduction of the acetone. This createdtechnical issues because the alkalinity of the primary amine degradedthe nitroprusside. Even when acids such as hydrochloric acid were usedto reduce the alkalinity and thus stabilize the nitroprusside,degradation still took place, albeit at a slower pace.

The method according to this aspect comprises disposing a reactant in areaction zone within the breath analysis device, wherein the reactantcomprises a primary amine disposed on a surface, and wherein thereaction zone has an optical characteristic that is at a referencelevel.

In this method, however, the nitroprusside is provided in the form of aliquid solution within the breath analysis device, but pre-storedseparately from the reactant. The method further comprises using thebreath analysis device to cause the breath to contact the reactant inthe reaction zone so that the acetone in the breath reacts with theprimary amine to form a reaction product and, after the reaction producthas been formed, using the breath analysis device to cause thenitroprusside solution to contact and react with the reaction productand to facilitate a change in the optical characteristic of the reactionzone relative to the reference level. The method also comprises usingthe breath analysis device to detect the change in the opticalcharacteristic to sense the acetone in the breath.

By separately pre-storing the nitroprusside, degradation is avoided.Moreover, by providing it as a liquid, it can be more easily dispensedinto the primary amine, and it can serve as, or double as, a solvent ordeveloper solution.

In presently preferred implementations of this method and embodiments ofrelated breath analysis devices, the primary amine is disposed on aplurality of silica gel beads. Small bead sizes, such as a sizedistribution between 100 and 270 mesh, are preferred so that sensitivityand bed efficiency are heightened.

In preferred implementations of the method, the primary amine componentis maintained in an alkaline environment, in the absence of any volatileacids. The primary amine bed may be quenched with a non-volatile acid,for example, but preferably, such as sulfuric acid.

The pre-stored liquid nitroprusside solution preferably is in anon-alkaline environment, and consist essentially of a non-alkalinesolution. Preferably there are not components in the nitroprussidesolution that have a base dissociation constant less than 6. Thepre-stored nitroprusside solution also preferably is quenched with anon-volatile acid, such as sulfuric acid. This quenching preferably isundertaken so that the liquid nitroprusside solution has a pH of lessthan 8, and more preferably below 7. To further avoid degradation, theliquid nitroprusside solution also preferably is stored in the absenceof ambient light. This may be achieved by providing the liquid in anampoule or other container that has an opaque outer coating so thatlight is excluded during pre-storage.

In accordance with still another aspect of the invention, a method isprovided for sensing acetone in breath using a breath analysis device.In this method, degradation of the nitroprusside is avoided bypre-storing a nitroprusside in a non-alkaline environment.

The method comprises disposing a reactant in a reaction zone within thebreath analysis device, wherein the reactant comprises a primary aminedisposed on a surface, and wherein the reaction zone has an opticalcharacteristic that is at a reference level. As noted, a nitroprussideis pre-stored in the breath analysis device in a non-alkalineenvironment. The method further comprises using the breath analysisdevice to cause the breath to contact the reactant in the reaction zoneso that the acetone in the breath reacts with the primary amine to forma reaction product. After the reaction product has been formed, themethod comprises using the breath analysis device to cause thenitroprusside to contact and react with the reaction product and tofacilitate a change in the optical characteristic of the reaction zonerelative to the reference level, and using the breath analysis device todetect the change in the optical characteristic and to sense the acetonein the breath.

The non-alkaline environment for the pre-stored nitroprusside accordingto this aspect of the invention, which preferably does not include anycomponents or substances with a base dissociation constant less than 6,can be achieved by quenching the primary amine on the surface with anon-volatile acid, e.g., such as sulfuric acid, so that the primaryamine on the surface has a pH that is less than 8, and more preferablyless than 7.

Given the different characteristics of the primary amine and thenitroprusside, the method preferably comprises pre-storing these twocomponents separately, preferably using a gas-tight barrier or othermeans of isolation.

The nitroprusside component may be in the form of a liquid, but it alsomay be in solid form. In a preferred implementation of the method, boththe primary amine and the nitroprusside are disposed on silica gel beadshaving a mesh size of about between 100 and 270. The beads areintermingled. A liquid developer solution is dispensed on theintermingled beads, which enables the reactions that give rise to thechange in optical characteristic.

Example 1

APTES beads were made by adding 0.5 grams (“g”) of 130 to 140 meshsilica gel to 200 microliters (“uL”) of APTES in 800 uL of propanol anddrying at 80° C. Following drying, the APTES beads were cured at 110° C.for one hour (“hr”). This was done to create the “first” surface.

DEAPMOS beads were made by adding 0.25 g of 130 to 140 mesh silica gelto 100 uL of DEAPMOS and 400 uL of propanol and drying as describedabove. Following drying, 0.25 g of the beads were added to 0.75 mL of 1normal (“N”) sulfuric acid (H2SO4) and 0.25 milliliters (“mL”) of diH2Oand incubated for 10 minutes (“min.”) while rocking. 0.1 g of sodiumnitroprusside (“SNP”) and 0.04 g of magnesium sulfate (MgSO4) weredissolved in the mixture, and then the beads were vacuum filtered anddried at 110° C. for 20 min. 0.25 g of the DEAPMOS/SNP beads were thenmixed with 0.5 g of APTES beads and packaged into separate aliquots.This was done to create the “second” surface.

The beads were packed into a ⅛ inch (“in.”) inside diameter Tygon tubingthat was 0.3 in. long. 0.5 parts per million (“ppm”) of acetone in drynitrogen gas (N2) was passed across the beads at 200 mL/min. for 3 min.Then 90 uL of developer solution (0.5% diethanolamine in 25%dimethylsulfoxide, 75% methanol) was added to the beads and the mixturewas allowed to incubate for 3 min. prior to imaging. When viewed at 3min., a short, but easily visible, color bar was observed.

The use of an acidic environment to create the “second” surface, andparticularly the use of an acid with a vapor pressure less than about 1atm at 22 C in its 99% pure form, provides significantly improvedstability and results over prior known approaches. If this method hadbeen implemented using HCl instead of sulfuric acid, the stability ofsodium nitroprusside would have been relatively low. The pH of theprimary amines would have also changed over time, reducing thereactivity of the primary amines. Although HCl (i.e., the acidreportedly used by Abbott) temporarily stabilizes the SNP, the HCl isextremely volatile. The tertiary amines eventually regain theiralkalinity to the degree that HCl is lost. This results in the rapiddegradation of SNP. Based on tests conducted by the inventors hereof,the method using HCl (0.5 g DEAPMOS beads manufactured with 1 mL 1 NHCl) fails in less than 12 hrs at 102° C. This is in contrast to themethod using H2SO4 (0.5 g DEAPMOS beads manufactured with 1 mL 1 NH2SO4), which lasts more than 4 times as long at 102° C.

Restricting the volatility as set forth herein above, e.g., using a lessvolatile acid than HCl, significantly improved results. In this specificexample, using H2SO4, the method quadrupled the stability to 48 hrs at102° C. over the HCl configuration reportedly used by Abbott, which isequivalent to 6 to 12 months at room temperature.

Because of the proton coupling to the tertiary amine, a strong positivecharge is present on the tertiary amine beads, making dissociation ofthe SNP very slow. Due to reversibility of the Schiff base formation,however, water should not be used. DMSO is added to help stabilize theSchiff base formation. Methanol does not have sufficient polarity tosolvate the nitroprusside in the presence of the positively-chargedtertiary amine. A weak base, such as diethanolamine, is preferred tohelp the protons diffuse, creating a charge gradient for thenitroprusside to follow. Also, once the nitroprusside is in solution, itstill must diffuse to the primary amines before the desired reactionwill take place. The kinetics of reaction with a tertiary amine adjustedto an appropriate pH level can require up to 10 min. for a completereaction at 0.5 ppm of acetone.

The sensitivity achieved with this preferred method implementation alsowas superior. Presumably this is due to a higher concentration ofnitroprusside than that used in the method disclosed by Abbott. Whilesubtle differences may exist, the overall results were pronounced. InExample 1, a breath sample containing 0.5 ppm of acetone yielded achange visible as a very short dark line following a lengthy developmenttime.

In summary, according to the Abbott patents, Abbott coupled SNP to atertiary amine synthesized from ethylamine and an epoxysilane. Theyadjusted the pH of the tertiary amine component to 4.5 using HCl.

Using the method as set forth in Example 1, the present inventorsquadrupled the stability (e.g., length of time that it is stable) byusing H2SO4 instead of HCl to pH adjust the tertiary amine. By using ahigher concentration of SNP the sensitivity and kinetics were enhanced.The SNP was also coupled to the beads with a different chemistry, i.e.,diethylaminopropyl trimethoxysilane, which provides the added benefit ofsimplifying manufacturing.

The Schiff base is capable of attacking the nitrosyl group of thenitroprusside under the right conditions. And in doing so, it forms acolored product that is visible to the eye.

Nitroprusside reportedly reacts with Schiff bases under alkalineconditions. The literature generally reports that the optimum pH isequal to the pKa of the conjugate acid, where 50% of the amino groupsforming the Schiff bases are protonated. For an aminopropyl silane, theoptimum pH is just over 10. In preferred embodiments and methods, the pHof the primary amines is adjusted between about 9 and about 11, betweenabout 10 and about 10.5. Lower pH values are believed to completelyprotonate the Schiff base, precluding it from reacting withnitroprusside. Higher pH's tend to hasten the degradation of the coloredproduct and have no positive charge to attract the nitroprusside with.In practice, we have observed the fastest kinetics for nitroprussidereaction with Schiff bases formed from aminopropyl silane near a pH of10. The nitroprusside does not visibly react under highly acidicconditions.

Since the kinetics of Schiff base formation are relatively fast, thereaction is rate limited primarily by the nitroprusside reaction withthe Schiff base. Hence, the kinetics are governed principally bynitroprusside concentration, pH and stability of Schiff base formation(e.g., the absence of water).

The stability of nitroprusside is one of the determinants of thestability of acetone tests using nitroprusside. Nucleophiles react withnitroprusside. Even weak nucleophiles such as water are capable ofdegrading nitroprusside.

In SNP/acetone reactions, low pH's are not used on the primary amines orin the developer solution because they prevent nitroprusside fromreacting with the Schiff base. Nitroprusside is greatly stabilized byacid. This is believed to be because it quenches any nucleophiles, thuspreventing degradation. When nitroprusside is dried or in solution withacid, it appears to be indefinitely stable in pH of 1, even in thepresence of nucleophiles. In practice, some stability is observed at apH of 7, but order of magnitude increases are observed at lower pH's.

Nitroprusside is generally considered to be unstable in solution.However, the inventors of the present invention have discovered that, inlow pH, nitroprusside is very stable in solution. But even at high pH,nitroprusside can be stabilized when present at high concentrations.This is believed to be because the nucleophiles present in solution areat a lower concentration than the nitroprusside and are quenched throughreaction with nitroprusside. At sufficiently large concentrations, thishas a negligible impact on the amount of active nitroprusside left insolution. In practice, the present inventors have put sufficientnitroprusside in solution to quench even 0.1 M NaOH. Nitroprussideappears indefinitely stable in solution with no nucleophiles presentwhen protected from light. Examples would be nitroprusside in diH2O orin a methanol/DMSO solution.

Nitroprusside is generally considered to be unstable in the presence ofprimary amines. However, the present inventors have discovered that whenprimary amines are quenched with sufficient acid to lower the pH,nitroprusside can become indefinitely stable in their presence.

When protected from light and the ambient, nitroprusside is stable forlong periods of time when dried onto surfaces with neutral pH andwithout nucleophiles. Examples would be silica gel or, less nucleophilicstill, teflon or polystyrene, or various other materials known to thoseskilled in the art.

The base serves to enhance solubility of the nitroprusside. Until thepositive charge on the surface is removed, very little nitroprusside issoluble, even in a mixture of 25% DMSO in MeOH over a period of 10minutes. Also, the base enables color formation. Until the protons areremoved from the surface, the nitroprusside remains in a completelyprotected form with no visible reaction. Where no pH change is necessaryand the nitroprusside, e.g., SNP, is not coupled to a positively chargedsurface, no base is necessary.

The DMSO has an unexpected benefit. The Abbott patents mention that DMSOwas added to “stabilize” color formation. In the single bead methoddescribed herein, a solution with APTES in methanol without DMSO appearsto wash acetone and color products downstream. In contrast, when DMSO isadded to the mix, the acetone/color product is more stable in theposition it starts in.

Performing the reaction in the single bead method described hereinwithout methanol (i.e., 62.5% DMSO, 37.5% APTES) yields no visiblereaction. Although some of the nitroprusside was clearly in solution,methanol may be required to assist with dissociation such thatnitroprusside becomes available for reaction. Also, substitution ofpropanol for methanol results in a uniform darkening of the reagent withor without the presence of acetone.

Holding DMSO constant at 25% and varying APTES concentration in methanolfrom 50%, 37.5%, 18.75%, and 9% showed that forward kinetics wereroughly equal for 18.75% and higher concentration, but began to slowonce APTES dropped to 9%. Destructive kinetics (i.e., loss of colorformation) and violent bubble production inhibiting a clear andconsistent view of the beads were substantially higher at 50% APTES. Forrapid kinetics and minimum destructive/bubble kinetics, an APTESconcentration of between 15 and 37% is preferred. However, lowerconcentrations, i.e., 10%, may be more suitable for smooth, consistentresults on longer columns or with larger amounts of developer solutionpassing across the beads.

In accordance with another aspect of the invention, a method is providedfor analyzing acetone in breath, wherein the method comprises providinga cavity, and locating within the cavity a primary amine disposed on afirst surface. The primary amine on the first surface comprises cavitycontents having a reference optical property. The method also comprisesproviding a nitroprusside in a nitroprusside solution initiallyseparated from the primary amine on the first surface, whereinconditions of the nitroprusside solution when separated from the primaryamine on the first surface are selected to stabilize the nitroprussiderelative to the reactivity of the nitroprusside in the cavity with theprimary amine. The method further comprises causing the breath to moveinto the cavity so that it contacts the primary amine to create aprimary amine reaction product, and causing the nitroprusside solutionto enter the cavity and the nitroprusside to react with at least one ofthe acetone and the primary amine reaction product, to cause orfacilitate a change in an optical property of the cavity contentsrelative to the reference optical property, and analyzing the breath forthe presence of the acetone using the change in the optical property.

A summary of the presently preferred implementation of this method isprovided in FIG. 32. A preferred but merely illustrative methodimplementation of this aspect of the invention is provided by thefollowing example.

Example 2

APTES beads were made by adding 0.5 g 130 to 140 mesh silica gel to 200uL APTES in 800 uL Propanol and drying at 80 C. Following drying, theAPTES beads were cured at 110° C. for 1 hr. 0.5 g of APTES beads wereadded to 0.4 mL 1 N H2SO4 and 0.6 mL propanol and dried at 110° C. for30 min.

Plain silica SNP beads were made by adding 0.3 g 130 to 140 mesh silicato 500 uL diH2O with 0.075 g SNP and 0.03 g MgSO4. Beads were dried at110° C. for about 45 min. Plain silica SNP beads were mixed with APTESbeads in a 1:2 ration and divided into aliquots

Beads were packed into a ⅛″ inside diameter column that was 0.3″ long.0.5 ppm acetone in dry N2 was passed across the beads at 200 mL/min for3 min. Then 90 uL of developer solution (25% dimethylsulfoxide, 75%methanol) was added to the beads and allowed to incubate for 3 min priorto imaging. A short, but easily visible, color bar was present afteronly 30 seconds.

A neutral or “plain” surface does not have nucleophiles present on it,such as tertiary amines, and do not generally require the addition ofextra acid. SNP is dried onto the surface and the surface is mixed witha second aminated surface, such as APTES beads.

The present inventors have used both silica and polystyrene as surfaces,but the potential embodiments are numerous. The polystyrene is inertwhereas the silica does have hydroxyl groups that have some nucleophilepotential.

The stability of SNP on either surface is believed to exceed that of theAbbott method. When acid is used, there is a problem with protondiffusion to the primary amines, changing their performance in a givendeveloper solution over time. When no acid is used, the nitroprusside isstable on silica gel for more than 96 hours at 102° C. That is more than8 times the stability seen in the reported Abbott method.

The neutral or plain surface has no net charge, so nitroprusside can besolvated with methanol on its own. However, DMSO helps the components gointo solution very quickly and stabilizes the Schiff base. Because ofdiffusion, the kinetics are not much better than the Abbott method.

The neutral surface method as described herein does not develop a pH orcharge gradient because protons are not used to stabilize or retain theSNP on the surface. However, as the SNP is solvated, it can form aslight gradient as it is washed away from the surface. Providedsufficient developer solution is used, this gradient appears minor forboth the methanol and DMSO/methanol developer solutions.

In accordance with another aspect of the invention, a method is providedfor analyzing acetone in breath, wherein the method comprises providinga cavity, and locating within the cavity a primary amine disposed on afirst surface. The primary amine on the first surface comprises cavitycontents having a reference optical property. The method also comprisesproviding a nitroprusside in a nitroprusside solution initiallyseparated from the primary amine on the first surface, whereinconditions of the nitroprusside solution when separated from the primaryamine on the first surface are selected to stabilize the nitroprussiderelative to the reactivity of the nitroprusside in the cavity with theprimary amine. The method further comprises causing the breath to moveinto the cavity so that it contacts the primary amine to create aprimary amine reaction product, and causing the nitroprusside solutionto enter the cavity and the nitroprusside to react with at least one ofthe acetone and the primary amine reaction product, to cause orfacilitate a change in an optical property of the cavity contentsrelative to the reference optical property, and analyzing the breath forthe presence of the acetone using the change in the optical property.

A preferred implementation of this method is provided in FIG. 33.

An anhydrous nitroprusside solution can be made by dissolving betweenabout 0.5 and 10%, between about 2 and 8%, between about 4 and 7% sodiumnitroprusside in an anhydrous, organic solvent. In some embodiments, theorganic solvent is an alcohol. In a preferred embodiment, the organicsolvent comprises methanol. In an even more preferred embodiment,dimethylsulfoxide is added as a stabilizer, between about 1 and 50%,between about 10 and 40%, between about 20 and 30%.

The following example provides an illustration of this aspect of theinvention.

Example 3

APTES beads were made by adding 0.5 g 130 to 140 mesh silica gel to 200uL APTES and 400 uL propanol. The beads were vortexed thoroughly for 10seconds. 0.4 mL 1 N H2SO4 was added and vortexed for 10 seconds. Thebeads were incubated at 80° C. for 10 min and then cured at 110° C. for1 hr.

1.67%, 2.5%, 5%, 6.67% and 10% SNP solutions were made by dissolving SNPin 25% DMSO in methanol. Solutions were stored in light proofcontainers.

The beads were packed into a ⅛″ inside diameter Tygon tube that was 0.3″long. 0.5 ppm acetone in dry N2 was passed across the beads at 200mL/min for 3 min. Then 90 uL of SNP solution was added to the beads andallowed to incubate for 3 min prior to imaging. A dark and easilyvisible color bar was present. The kinetics of the reaction were fasterfor higher concentrations of SNP. 10% SNP formed a precipitate afterseveral days at standard temperature. The 6.67% SNP solution was stablefor more than two weeks at room temperature, in the freezer and at 61 C.

A 150 uL 6.67% SNP solution was placed in a 5/16 inch outside diameterby ½ A inch long black polyethylene tube and heat sealed on either sidewith 5/16 inch diameter mylar foil coated with polyethylene.

A ¼ inch porous polyethylene disk is placed in one end of the cartridgeand the ampoule containing the SNP solution is placed in after it. A5/16 inch absorbent glass wool disk was placed on top of the ampoule andthe top of the cartridge was sealed shut with a layer of mylar foil.

The cartridge was then flipped over and APTES beads (from the SNPSolution method example herein above) were placed into the middleportion of the cartridge. Another ¼ inch porous polyethylene disk wasused to trap the APTES beads in place. Then 30 to 60 mesh CaCl2 wasadded. Then, another 5/16 inch piece of glass wool was placed in thecartridge and the bottom was sealed shut with a layer of mylar foil.

The kinetics for SNP in solution are very rapid when SNP is at aconcentration greater than 2% and when used with primary amines with apH of between about 10 and 10.5.

SNP in solution has a larger gradient if acid is present in thedeveloper solution or on the surface of the beads. This is because SNPis extracted from solution by positively-charged surfaces, created byproton coupling with primary amines. However, using a larger amount ofsolution can overcome these problems.

In accordance with yet another aspect of the invention, a method isprovided for analyzing acetone in breath. The method comprises preparinga surface upon which is disposed a primary amine and a nitroprusside.The preparation comprises disposing the primary amine and thenitroprusside in an acidic environment. The method also compriseslocating the surface upon which is disposed the primary amine and thenitroprusside within a cavity. The surface comprising the primary amineand the nitroprusside comprises a reference optical property. Thesurface preferably is in proximate contact with a solution. The methodfurther comprises causing the breath to move into the cavity so that itcontacts the primary amine and the nitroprusside to cause or facilitatea change in an optical property of the surface relative to the referenceoptical property. The method further comprises analyzing the breath forthe presence of the acetone using the change in the optical property.

A preferred implementation of the method is provided in FIG. 34.

The present inventors have discovered that a truly stable SNP/primaryamine group can be obtained by adjusting the pH to less than about 6 andeven as low as 1. This results in a stability that exceeds that reportedby Abbott, and that of urinalysis chemistry.

The urinalysis groups cannot use a pH of less than 7, because it makesthe SNP non-reactive to acetone and other ketones. The present inventorshave discovered that, by adding a base at least as strong as the primaryamine to the developer solution at high concentration, superiorstability can be achieved. This is believed to remove the protons,changing the pH and allowing the reaction to proceed.

The pH of the primary amine preferably is adjusted to between about 0and 6, more preferably between about 0.5 and 4, and even more preferablybetween about 1 and 2. Nitroprusside is added to the acidic primaryamines preferably between about 1 and 20%, more preferably between about5 and 15%, and even more preferably between about 8 and 12%. Thesolution is then vacuum filtered and the beads are dried. In someembodiments, the beads are dried under vacuum. In other embodiments,they are dried on heat. In a preferred embodiment, they are dried at110° C. for 30 min.

The developer solution comprises an organic solvent and a base. Thesolution preferably is between about 5 and 50%, more preferably betweenabout 10 and 40%, and still more preferably between about 20 and 30%volume/volume composition with a base that has a pKb less than or equalto the pKb of the primary amine. The base preferably is aminopropyltriethoxysilane. More preferably, a base is used that is 1 or 2 unitsless than the pKb of the primary amine on the surface.

Example 4

APTES beads were made by adding 0.5 g of 130 to 140 mesh silica gel to200 uL of APTES in 800 uL of propanol and drying at 80° C. Followingdrying, the APTES beads were cured at 110° C.

0.5 g of the APTES beads thus made were added to 1.5 mL of 1N HCl and0.5 mL of H2O and incubated for 10 min. while rocking. 0.2 g of SNP wasadded and dissolved. The beads were filtered under vacuum, and thendried at 110° C. for 20 min.

The beads were packed into a ⅛″ Inside diameter column that was 0.3″long. 0.5 ppm of acetone in dry N2 was passed across the beads at 200mL/min for 3 min. Then 90 uL of developer solution (25%dimethylsulfoxide, 37.5% APTES in methanol) was added to the beads andthey were allowed to incubate for 3 min. prior to imaging. A dark andeasily visible color bar was observed.

This yielded a very stable method at 102° C. In this method, the primaryamine was neutralized with acid (HCl, but less volatile acids arepreferred, such as H2SO4) and coupled the SNP directly to it. Becausethe protons are already coupled to the primary amine, there is nodiffusion over time that can lead to a change in performance. Also,because the nucleophiles are quenched and form a salt with thenitroprusside, the nitroprusside is stable over time. This method hasshown stability for more than 96 hrs at 102° C. (i.e., estimatedstability in excess of 1 yr at standard or room temperature).

Implementations of this method yield relatively fast kinetics. Kineticsof reaction are driven by the concentration of Schiff base, theconcentration of fully-solubilized nitroprusside, the diffusion time fornitroprusside, and the pH. The kinetics are advanced by the relativelyhigh concentration of primary amines and nitroprusside, the fact thatdiffusion is negligible with nitroprusside coupled directly to theprimary amines, and with the proper reaction conditions.

The method can present problems, however, with gradient formation. Inorder to get the primary amines to react with acetone/SNP to form thecolor change, the pH must be greatly increased. This requires the use ofstrong bases that can strip the proton away from the primary amine. Thestrong bases are then quenched from the protons they strip from thesurface, creating a pH gradient and a charge gradient along the column.The longer the column relative to the diffusion time of the developersolution, the more of a problem this is. Further, the charge gradientcauses a larger amount of nitroprusside to follow the positive charges,creating a color gradient. The very high pH at the beginning of thecolumn where the developer solution first comes in contact is sufficientto completely bleach the signal.

This problem can be addressed by using a short column, a pressurizedmethod to add the developer solution, or a method to introduce thedeveloper solution throughout the length of the column simultaneously.

It will be appreciated that the invention is not limited to the specificembodiments and method implementations described herein. The descriptionherein has largely been explained with respect to human patients orsubjects, but this is not necessarily limiting. The principles of theinvention also may be applied in veterinary applications.

Having now described the invention and preferred embodiments and methodsof it, it will be appreciated that the invention in its broader aspectsis not limited to the specific details, representative devices andmethods, and illustrative examples shown and described herein above. Asnoted herein above, the invention according to its various aspects isparticularly pointed out and distinctly claimed in the attached claimsread in view of this specification, and appropriate equivalents.

We claim:
 1. A method for sensing acetone in breath using a breathanalysis device, the method comprising: providing a breath analysisdevice having a reactant in a reaction zone, wherein the reactantcomprises a primary amine disposed on a surface, and wherein thereaction zone has an optical characteristic that is at a referencelevel; a liquid nitroprusside solution disposed within the breathanalysis device separately from the reactant; using the breath analysisdevice to cause the breath to contact the reactant in the reaction zoneso that the acetone in the breath reacts with the reactant to form areaction product; after the reaction product has been formed, using thebreath analysis device to cause the nitroprusside solution to contactand react with the reaction product and to facilitate a change in theoptical characteristic of the reaction zone relative to the referencelevel; and using the breath analysis device to detect the change in theoptical characteristic to sense the acetone in the breath.
 2. A methodas recited in claim 1 wherein causing the nitroprusside solution tocontact the reaction product has an optical characteristic that is at asecond reference level, and further wherein causing the nitroprussidesolution to react with the reaction product facilitates a change in theoptical characteristic of the reaction zone relative to the secondreference level.
 3. A method as recited in claim 1, wherein the surfacecomprises a silica gel.
 4. A method as recited in claim 1, wherein thesurface comprises a plurality of silica gel beads.
 5. The method asrecited in claim 4, wherein the silica gel beads have a sizedistribution between 270 and 100 mesh.
 6. A method as recited in claim1, wherein the providing a breath analysis device having a reactant inthe reaction zone step comprises providing a device in which thereactant is maintained in an alkaline environment.
 7. A method asrecited in claim 1, wherein the providing a breath analysis devicehaving a reactant in the reaction zone step comprises providing a devicein which the reactant is maintained in the absence of volatile acid. 8.A method as recited in claim 1, wherein the providing a breath analysisdevice having a reactant in the reaction zone step comprises providing adevice in which the primary amine is quenched with a non-volatile acid.9. A method as recited in claim 1, wherein the providing a breathanalysis device having a reactant in the reaction zone step comprisesproviding a device in which the primary amine is quenched with sulfuricacid.
 10. A method as recited in claim 1, wherein the liquidnitroprusside solution within the breath analysis device consistsessentially of a non-alkaline solution.
 11. A method as recited in claim1, wherein the liquid nitroprusside solution does not include asubstance with a base dissociation constant less than
 6. 12. A method asrecited in claim 1, comprising quenching the liquid nitroprussidesolution so that the liquid nitroprusside solution has a pH of less than8.
 13. A method as recited in claim 1, comprising, quenching the liquidnitroprusside solution so that the liquid nitroprusside solution has apH of less than
 7. 14. A method as recited in claim 1 wherein the liquidnitroprusside solution is stored in the absence of ambient light.
 15. Amethod for sensing acetone in breath using a breath analysis device, themethod comprising: providing a breath analysis device comprising areactant in a reaction zone within the breath analysis device, whereinthe reactant comprises a primary amine disposed on a surface, andwherein the reaction zone has an optical characteristic that is at areference level; and a liquid nitroprusside solution in the breathanalysis device in a non-alkaline environment; using the breath analysisdevice to cause the breath to contact the reactant in the reaction zoneso that the acetone in the breath reacts with the primary amine to forma reaction product; after the reaction product has been formed, usingthe breath analysis device to cause the nitroprusside to contact andreact with the reaction product and to facilitate a change in theoptical characteristic of the reaction zone relative to the referencelevel; and using the breath analysis device to detect the change in theoptical characteristic and to sense the acetone in the breath.
 16. Amethod as recited in claim 15, comprising quenching the primary amine onthe surface with a non-volatile acid so that the primary amine on thesurface has a pH that is less than
 8. 17. A method as recited in claim15, comprising quenching the primary amine on the surface with anon-volatile acid so that the primary amine on the surface has a pH thatis less than
 7. 18. A method as recited in claim 16, wherein thenon-volatile acid comprises sulfuric acid.
 19. A method as recited inclaim 15, comprising pre-storing the nitroprusside separately from thereactant prior to the causing of the nitroprusside to contact theprimary amine reaction product step.
 20. A method as recited in claim19, wherein the pre-storing of the nitroprusside separately from thereactant comprises using a gas-tight barrier.
 21. A method as recited inclaim 15, wherein the nitroprusside solution is stored in the absence ofa substance with a base dissociation constant less than
 6. 22. A methodas recited in claim 15, wherein the nitroprusside is quenched with anon-volatile acid.
 23. A method as recited in claim 22, wherein thenon-volatile acid comprises sulfuric acid.
 24. A method as recited inclaim 15, wherein the nitroprusside solution is quenched with anon-volatile acid so that the environment has a pH is below
 8. 25. Amethod as recited in claim 15, wherein the nitroprusside is quenchedwith a non-volatile acid so that the environment has a pH is below 7.26. A method as recited in claim 15, wherein: the surface upon which theprimary amine is disposed comprises a first surface; the nitroprussideis disposed on a second surface other than the first surface; the firstand second surfaces are intermingled prior to the causing of thenitroprusside to contact the reaction product; and the causing of thenitroprusside to contact and react with the reaction product comprisesdispensing a liquid developer solution to contact the first and secondsurfaces.
 27. A method as recited in claim 15, wherein: thenitroprusside is coupled to the reactant prior to the causing of thenitroprusside to contact the primary amine product; the reactant and thenitroprusside are disposed in the non-alkaline environment; and thecausing of the nitroprusside to contact and react with the reactionproduct comprises dispensing a liquid developer solution to contact thenitroprusside and the reaction product.